VARIANTS OF HUMANIZED ANTI-CARCINOMA MAb CC49

ABSTRACT

The invention is directed towards mouse-human chimeric variants of CC49 monoclonal antibodies with minimal murine content. A first aspect of the invention provides CDR variants of humanized monoclonal antibody (HuCC49) in which less than all six (three heavy chain and three light chain) Complementarity Determining Regions (CDRs) of CC49 are present. A second aspect of the invention provides SDR variants of humanized monoclonal antibody (HuCC49) in which only Specificity Determining Regions (SDRs) of at least one CDR from CC49 are present. The invention is also directed towards biotechnological methods of making the variants and therapeutic methods of using the variants.

PRIORITY CLAIM

This is a division of U.S. patent application Ser. No. 10/927,433, filedAug. 25, 2004, which is a division of U.S. patent application Ser. No.09/830,748, filed Apr. 30, 2001, now U.S. Pat. No. 6,818,749, issuedNov. 16, 2004, which is the §371 U.S. national stage of InternationalApplication No. PCT/US99/25552 filed Oct. 29, 1999, which was publishedin English under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 60/106,534 filed Oct. 31, 1998 and U.S.Provisional Application No. 60/106,757 filed Nov. 2, 1998, all of whichare incorporated herein in their entirety.

BACKGROUND

Antibodies are specific immunoglobulin (Ig) polypeptides produced by thevertebrate immune system in response to challenges by foreign proteins,glycoproteins, cells, or other antigenic foreign substances. The bindingspecificity of such polypeptides to a particular antigen is highlyrefined, with each antibody being almost exclusively directed to theparticular antigen which elicited it. This specificity resides in thestructural complementarity between the antibody combining site and theantigenic determinant. Antibody combining sites are made up of residuesthat are primarily from the hypervariable or complementarity determiningregions (CDRs). Occasionally, residues from nonhypervariable orframework regions do influence the overall domain structure and hencethe combining site.

There are two major methods for generating vertebrate antibodies:generation of polyclonal antibodies in situ by mammalian B lymphocytesand generation of monoclonal antibodies in cell culture by B cellhybrids.

To generate antibodies in situ, an animal (such as a mouse or rabbit) isinjected with an antigen. Several weeks later, blood is drawn from theanimal and centrifuged. The resulting serum contains antibodies againstthe injected antigen. The resulting antibodies are polyclonal antibodiesbecause they are products of many different populations of antibodyproducing cells and hence differ somewhat in their precise specificityand affinity for the antigen.

Monoclonal antibodies are produced using hybridoma technology in whichan antibody producing cell is fused with a tumor cell that has thecapacity for unlimited proliferation. In contrast to polyclonalantibodies, monoclonal antibodies are homogeneous because they aresynthesized by a population of identical cells that are derived from asingle hybridoma cell.

However, the use of monoclonal antibodies in humans is severelyrestricted when the monoclonal antibody is produced in a non-humananimal. Repeated injections in humans of a “foreign” antibody, such as amouse antibody, may lead to harmful hypersensitivity reactions, i.e.,anti-mouse antibody (HAMA) or an anti-idiotypic, response. The HAMAresponse makes repeated administrations ineffective due to an increasedrate of clearance from the patient's serum and/or allergic reactions bythe patient.

Attempts have been made to manufacture human-derived monoclonalantibodies using human hybridomas. Unfortunately, yields of monoclonalantibodies from human hybridoma cell lines are relatively low comparedto mouse hybridomas. Additionally, human cell lines expressingimmunoglobulins are relatively unstable compared to mouse cell lines,and the antibody producing capability of these human cell lines istransient. Thus, while human immunoglobulins are highly desirable, humanhybridoma techniques have not yet reached the stage where humanmonoclonal antibodies with the required antigenic specificities can beeasily obtained.

Thus, antibodies of non-human origin have been genetically engineered tocreate chimeric or humanized antibodies. Such genetic engineeringresults in antibodies with a reduced risk of a HAMA response compared tothat expected after injection of a human patient with a mouse antibody.For example, chimeric antibodies can be formed by grafting non-humanvariable regions to human constant regions. Khazaeli et al. (1991), J.Immunotherapy 15:42-52. Generally humanized antibodies, are formed bygrafting non-human complementarity determining regions (CDRs) onto humanframework regions (FRs) (See European Patent Application 0 239 400;Jones et al. (1986), Nature (London), 321:522-525; and Reichman et al.(1988), Nature (London), 332:323-327). Typically, humanized monoclonalantibodies are formed by grafting all six (three light chain and threeheavy chain) CDRs from a non-human antibody into Framework Regions (FRs)of a human antibody. Alternately, Fv antibodies (See U.S. Pat. No.4,642,334) or single chain Fv (SCFV) antibodies (See U.S. Pat. No.4,946,778) can be employed to reduce the risk of a HAMA response.

However, these modified antibodies still retain various non-human lightand heavy chain variable regions: the chimeric, Fv and single chain Fvantibodies retain entire non-human variable regions and CDR-graftedantibodies retain CDR of non-human origin. Such non-human regions canelicit an immunogenic reaction when administered to a human patient.Thus, many humanized MAbs remain immunogenic in both subhuman primatesand in humans, with the humoral response of the host directed towardsthe variable region of these MAb (Hakimi et al. (1991), J. Immunol.,147:1352-1359; Stephens et al. (1995), Immunology, 85:668-674; Singer etal. (1993), J. Immunol., 150:2844-2857; and Sharkey et al. (1995),Cancer Res. 55:5935s-5945s).

One known human carcinoma tumor antigen is tumor associatedglycoprotein-72 (TAG-72), as defined by monoclonal antibody B72.3 (SeeThor et al., (1986) Cancer Res., 46:3118-3124; and Johnson et al.,(1986), Cancer Res., 46:850-85). TAG-72 is associated with the surfaceof certain tumor cells of human origin.

Numerous murine monoclonal antibodies have been developed which havebinding specificity for TAG-72. Exemplary murine monoclonal antibodiesinclude the “CC” (colon cancer) monoclonal antibodies, which are alibrary of murine monoclonal antibodies developed using TAG-72. CertainCC antibodies have been deposited with the ATCC, including CC49 (ATCCNo. HB 9459). Monoclonal antibody (MAb) CC49 is a second-generationantibody of B72.3 that reacts with the pancarcinoma tumor-associatedantigen, TAG-72. Radiolabeled MAb CC49 has been shown to target tumor inboth animal models and in ongoing radioimmunotherapeutic andraiodimmunodiagnostic clinical trials. (Divgi et al. (1994) Nucl. Med.Biol., 21:9-15; Meredith et al. (1994), J. Nucl. Med., 35:1017-1022;Mulligan et al. (1995), Clin. Cancer Res., 1:1447-1454; Arnold et al.(1992), Ann. Surgery, 216:627-632)The potential clinical utility of MAbCC49 is evident both from animal studies and ongoing clinical trialswith the antibody. However, patients administered MAb CC49 do generateHAMA responses (Divgi et al, (1994) Nuc. Med. Biol., 21:9-15); Mulliganet al., (1995) Clin. Cancer Res., 1:1447-1454).

A humanized monoclonal antibody (HuCC49) has been formed by graftinghypervariable regions from monoclonal antibody CC49 into variable light(V_(L)) and variable heavy (V_(H)) frameworks of human monoclonalantibodies LEN and 21/28′CL, respectively, while retaining murineframework residues required for integrity of the antigen combining-sitestructure. (See, Kashmiri et al., (1995) Hybridoma, 14(5):461-473). ThisHuCC49 was shown to bind the TAG-72 antigen, albeit with a loweraffinity, and demonstrated equivalent tumor targeting in animal modelsbearing human tumor xenografts.

It has been shown that not all residues of CDRs are critical in thecomplementarity of antigen/antibody surfaces. Known structures of theantigen-antibody complexes suggests that only 20-33% of CDR residues areinvolved in antigen contact (Padlan, (1994) Mol. Immunol., 31: 169-217).A comprehensive analysis of the available data of the sequences and thethree dimensional structure of antibody combining sites has helpedidentify CDR residues that may be most critical in the antigen antibodyinteraction (Padlan et al., (1995) FASEB J., 9:133-139). These residuesare designated as specificity determining residues (SDRs). Specificitydetermining residues vary between antibodies.

SUMMARY

The invention is directed towards mouse-human chimeric variants of CC49monoclonal antibodies with minimal murine content which elicit minimaladverse responses when administered to a human patient. The invention isalso directed towards biotechnological methods of making the variantsand therapeutic methods of using the variants.

A first aspect of the invention provides CDR variants of humanizedmonoclonal antibody (HuCC49) in which less than all six (three heavychain and three light chain) Complementarity Determining Regions (CDRs)of CC49 are present. A second aspect of the invention provides SDRvariants of humanized monoclonal antibody (HuCC49) in which onlySpecificity Determining Regions (SDRs) of at least one CDR from CC49 arepresent. Surprisingly, the CC49 variants of the invention have the sameor similar binding affinity as humanized CC49 monoclonal antibody whichincludes all six (three heavy chain and three light chain) CDRs.

In particular, the invention relates to variants of HuCC49 in whicheither L-CDR1 or L-CDR2, or both, are from a human monoclonal antibody(LEN) (LEN L-CDR1 and LEN L-CDR2 correspond to SEQ ID NOs: 7 and 8,respectively). These variants of HuCC49 have the substantially the sameaffinity constant as HuCC49, or show only a two fold lower relativeaffinity than that of HuCC49.

Other suitable variants include corresponding human residues at position97 of the light chain in addition to a substitution of L-CDR1 and/orL-CDR2 from CC49 with the corresponding CDRs from a human antibody. Inanother embodiment, the variant includes a substitution at position 97on the light chain in addition to a substitution of L-CDR1 and/or L-CDR2from CC49 with the corresponding CDRs from a human antibody incombination with substitutions at positions 60, 61, 62 and 64 on theheavy chain. In another embodiment, the variant includes a substitutionat position 97 on the light chain in combination with substitutions atpositions 60, 61, 62 and 64 on the heavy chain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a basic immunoglobulin structure.

FIG. 2 shows a comparison of the CDR sequences of murine MAb CC49 andhumanized MAb HuCC49 with the corresponding CDR sequences of human MAbsLEN and 21/28′CL. Amino acid residues are numbered using the conventionof Kabat et al. The underlined numbers indicate the specificitydetermining residues (SDRs). CDR1, CDR2 and CDR3 within the light chainof HuCC49 and CC49 correspond to SEQ ID NOs: 1-3, respectively. CDR1,CDR2 and CDR3 within the heavy chain of HuCC49 and CC49 correspond toSEQ ID NOs: 4-6, respectively. CDR1, CDR2 and CDR3 within the lightchain of human antibody LEN correspond to SEQ ID NOs: 7-9, respectively.CDR1, CDR2 and CDR3 within the heavy chain of human antibody 21/28°CLcorrespond to SEQ ID NOs: 10-12, respectively.

FIG. 3 is a schematic representation of the eukaryotic expressionconstructs of the humanized heavy (B) chains of HuCC49. Thin linesrepresent sequences derived from the prokaryotic vectors pBR322,pBluescript SK⁻, or pCR II. Thick lines depict human K constant region.Boxes with vertical, horizontal, or cross bars show neomycin,mycophenolic acid, or hygromycin resistance genes; thin arrows showtheir transcriptional direction. Empty boxes are retroviral longterminal repeats, while thick arrows show the HCMV promoter and itstranscriptional direction. Only relevant enzyme sites are shown. A:ApaI; B: BamHI; C: ClaI; Hd: HindIII; Hp: HpaI; N: NheI; R: EcoRI; andS: SacII.

FIG. 4 is a schematic representation of the eukaryotic expressionconstructs of the humanized light chains of HuCC49. As with FIG. 3, thinlines represent sequences derived from the prokaryotic vectors pBR322,pBluescript SK⁺, or pCR II. Thick lines depict human k constant region.Boxes with vertical, horizontal, or cross bars show neomycin,mycophenolic acid, or hygromycin resistance genes; thin arrows showtheir transcriptional direction. Empty boxes are retroviral longterminal repeats, while thick arrows show the HCMV promoter and itstranscriptional direction. Only relevant enzyme sites are shown. A:ApaI; B: BamHI; C: ClaI; Hd: HindIII; Hp: HpaI; N: NheI; R: EcoRI; andS: SacII.

FIG. 5 is a schematic representation of the dual expression constructsof the variant heavy (H) and light (L) chain genes derived from thebaculovirus vector pAcUW51. P10 and polh represent p10 and polyhedrinpromoter; arrows show their direction of transcription. Ori and fl areSV40 and f1 origin of replication. Amp^(R) represents an ampicillinresistant gene.

FIG. 6 shows an SDS-PAGE analysis of purified MAb HuCC49 and itsvariants. All samples are shown in a reduced condition. Lane 1:molecular weight marker (Gibco Brl); Lanes 2-8: variants L-1, L-2, L-3,L-1,2, H-1, H-2 and H-3; Lane 9: HuCC49.

FIG. 7 shows an analysis of parental and variant forms of HuCC49 in acompetitive RIA. The antigen binding of the light chain (A) and heavychain (B) CDR variants was assessed using ¹²⁵I-labeled HuCC49. In panelA, the competitors were: HuCC49, L-1, L-2, L-3, L-1,2. In panel B, thecompetitors were: H-1, H-2 and H-3.

FIG. 8 shows the effect of light chain CDRs on binding of anti-idiotypicMAbs. The HuCC49 CDR variants were characterized in a competition RIAwith ¹²⁵I-HuCC49 and CC49 anti-idiotypic MAbs AI49-3 (panel A), AI49-1(panel B) and AI49-8 (panel C). The competitors were: HuCC49, L-1, L-2,L-3, L-1,2.

FIG. 9 shows the effect of heavy chain CDRs on binding of anti-idiotypicMAbs. The HuCC49 CDR variants were characterized in a competition RIAwith ¹²⁵I-CC49 and CC49 anti-idiotypic MAbs AI49-3 (panel A), AI49-1(panel B) and AI49-8 (panel C). The competitors were: HuCC49, H-1, H-2,H-3.

FIG. 10 shows an analysis of human anti-idiotypic antibodies to HuCC49variants using a competative RIA by HPLC methodology. A patient'santi-idiotypic response to CC49 was characterized using purifiedparental HuCC49 and CDR variants as competitors with ¹²⁵I-HuCC49. Theinability of a variant to inhibit complex formation of the patient'ssera with the ¹²⁵I-HuCC49 indicates that the CDR replaced from thevariant was immunogenic to the patient. In panel A, the competitorswere: HuCC49, L-1, L-2, L-3, L-1,2. In panel B, the competitors were:H-1, H-2 and H-3.

FIG. 11 shows the amino acid sequences of V_(L) frameworks of human MAbLEN (SEQ ID NOs: 33-36) and humanized V_(L) of CC49 (HuCC49) (SEQ ID NO:13) in panel A. Panel B shows the amino acid sequences of V_(H)frameworks of human MAb 21/28′CL (SEQ ID NOs: 37-40) and humanized V_(H)of CC49 (HuCC49) (SEQ ID NO: 14). Framework residues that are deemed tobe important in maintaining the combining site structure of CC49 aremarked by an asterisk.

FIG. 12 shows the nucleotide sequence of HuCC49 variable light (V_(L))(SEQ ID NO: 41 and SEQ ID NO: 42) and variable heavy (V_(H)) region (SEQID NO: 43 and SEQ ID NO: 44) genes in panels A and B, respectively.Sequences of flanking oligomers that do not encode the variable regiondomains or their leader peptides are shown in lowercase letters. TheV_(L) region (A) is encoded by nucleotides from positions 74 to 412,while nucleotides from position 70 to 415 (B) comprise the V_(H) region.The four overlapping oligomers depicted by long arrows in FIG. 12A arerepresented by SEQ ID NOs: 19-22. The four overlapping oligomersdepicted by long arrows in FIG. 12B are represented by SEQ ID NOs:15-18.

FIG. 13 is a graph of the results of a competition assay using variantsof HuCC49.

FIG. 14 shows the results of an HPLC analysis of patient reactivity tovariants of HuCC49. Competitors were at 5 Tg per reaction. The valuesare the percent of complexes, the higher molecular weight species,resolved by size-exclusion chromatography. Complex formation indicatesremoval of the epitope recognized by the patient's antibody. Inhibitionof complex formation indicates that the immunogenic epitope is stillpresent in the HuCC49 variant.

FIG. 15 is a graph showing the comparison of patient reactivity withHuCC49 and various variants thereof.

FIG. 16 is graph showing the immunoreactivity of variant⁹⁷L_(1,2)/^(60-62,64)H.

FIG. 17 is a graph of the pharmacokinetics of plasma retention of HuCC49and a variant thereof.

FIG. 18 is a table showing the biodistribution of i.v. administeredradiolabeled HuCC49 and variants in athymic mice bearing LS-174T humancolon carcinoma xenografts. Athymic mice bearing LS-174T human coloncarcinoma xenografts (s.c.) were coinjected with 1.4TCi of ¹³¹I-HuCC49and 4.4 of ¹²⁵I-Variant. The mice were sacrificed at the timepointsindicated, the organs harvested, wet-weighed and the radioactivitydetected in a K-scintillation counter. The percent weight injected doseper gram for each tissue was calculated. The standard error of the meanwas also calculated and were 0.06% ID/g or less.

FIG. 19. HPLC analysis of patient HAMA following intravenous injectionof ¹⁷⁷LU-CC49.

FIG. 20. HPLC analysis of patient' humoral response to the variableregion of MAb CC49. The percent complex formation has been plottedversus time for (solid lines) patients DS (◯), LW (□), JJ (Δ), DG (●),LJ (▪), TD(▴); (dotted lines) JG (◯), RW (□), JM A), EA (●), CP (▪), LQ(▴);

FIG. 21. Detection of patient anti-idiotypic antibody response to murineCC49.

FIG. 22. HPLC analysis demonstrating CDR specificity of patient LQ.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases and three letter code for amino acids, as defined in 37 C.F.R.1.822. In the accompanying sequence listing:

SEQ ID NO: 1 shows the amino acid sequence of CDR1 from the light chainof murine antibody CC49 and humanized antibody HuCC49.

SEQ ID NO: 2 shows the amino acid sequence of CDR2 from the light chainof murine antibody CC49 and humanized antibody HuCC49.

SEQ ID NO: 3 shows the amino acid sequence of CDR3 from the light chainof murine antibody CC49 and humanized antibody HuCC49.

SEQ ID NO: 4 shows the amino acid sequence of CDR1 from the heavy chainof murine antibody CC49 and humanized antibody HuCC49.

SEQ ID NO: 5 shows the amino acid sequence of CDR2 from the heavy chainof murine antibody CC49 and humanized antibody HuCC49.

SEQ ID NO: 6 shows the amino acid sequence of CDR3 from the heavy chainof murine antibody CC49 and humanized antibody HuCC49.

SEQ ID NO: 7 shows the amino acid sequence of CDR1 from the light chainof human antibody LEN

SEQ ID NO: 8 shows the amino acid sequence of CDR2 from the light chainof human antibody LEN

SEQ ID NO: 9 shows the amino acid sequence of CDR3 from the light chainof human antibody LEN

SEQ ID NO: 10 shows the amino acid sequence of CDR1 from the heavy chainof human antibody 21/28′CL

SEQ ID NO: 11 shows the amino acid sequence of CDR2 from the heavy chainof human antibody 21/28′CL

SEQ ID NO: 12 shows the amino acid sequence of CDR3 from the heavy chainof human antibody 21/28′CL

SEQ ID NO: 13 shows amino acid sequence of the V_(L) domain of thehumanized murine antibody HuCC49.

SEQ ID NO: 14 shows amino acid sequence of the V_(H) domain of thehumanized murine antibody HuCC49.

SEQ ID NOs: 15-32 show several synthetic oligonucleotides useful asprobes and/or primers.

SEQ ID NOs: 33-36 show amino acid sequences of consecutive portions ofthe framework of the V_(L) domain of the human antibody LEN.

SEQ ID NOs: 37-40 shows amino acid sequences of consecutive portions ofthe framework of the V_(H) domain of the human antibody 21/28′CL.

SEQ ID NO: 41 show the nucleic acid sequence including the V_(L) domainof the humanized murine antibody HuCC49 together with flankingoligomers.

SEQ ID NO: 42 shows the nucleic acid sequence complementary to SEQ IDNO: 41.

SEQ ID NO: 43 shows the nucleic acid sequence including the V_(H) domainof the humanized murine antibody HuCC49 together with flankingoligomers.

SEQ ID NO: 44 shows the nucleic acid sequence complementary to SEQ IDNO: 43.

DEFINITIONS

Prior to setting forth the invention, definitions of certain terms whichare used in this disclosure are set forth below:

Antibody: This refers to single chain, two-chain, and multi-chainproteins and glycoproteins belonging to the classes of polyclonal,monoclonal, chimeric and hetero immunoglobulins (monoclonal antibodiesbeing preferred); it also includes synthetic and genetically engineeredvariants of these immunoglobulins. “Antibody fragment” includes Fab,Fab′, F(ab′)₂, and Fv fragments, as well as any portion of an antibodyhaving specificity toward a desired target epitope or epitopes.

Chimeric antibody: This refers to an antibody which includes sequencesderived from two different antibodies, which typically are of differentspecies. Most typically, chimeric antibodies include human and murineantibody fragments, generally human constant and murine variableregions.

Humanized antibody: This refers to an antibody derived from a non-humanantibody, typically murine, and a human antibody which retains orsubstantially retains the antigen-binding properties of the parentantibody but which is less immunogenic in humans.

Complementarity Determining Region, or CDR: This refers to amino acidsequences which together define the binding affinity and specificity ofthe natural Fv region of a native immunoglobulin binding site. The lightand heavy chains of an immunoglobulin each have three CDRs, designatedL-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Bydefinition, the CDRs of the light chain are bounded by the residues atpositions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3);the CDRs of the heavy chain are bounded by the residues at positions 31and 35b (H-CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using thenumbering convention delineated by Kabat et al., (1991) Sequences ofProteins of Immunological Interest, 5th Edition, Department of Healthand Human Services, Public Health Service, National Institutes ofHealth, Bethesda (NIH Publication No. 91-3242).

Framework Region: This refers to amino acid sequences interposed betweenCDRs. These portions of the antibody serve to hold the CDRs in anappropriate orientation for antigen binding.

Specificity Determining Residue, or SDR: This refers to amino acidresidues of an immunoglobulin that are directly involved in antigencontact.

Constant Region: This refers to the portion of the antibody moleculewhich confers effector functions. In the present invention, the variantantibodies include constant regions derived from human immunoglobulins.The heavy chain constant region can be selected from any of fiveisotypes: alpha, delta, epsilon, gamma or mu. Heavy chains of varioussubclasses (such as the IgG subclass of heavy chains) are responsiblefor different effector functions. Thus, by choosing the desired heavychain constant region, humanized antibodies with the desired effectorfunction can be produced. The light chain constant region can be of thekappa or lambda type, preferably the kappa type.

Mammals: This refers to animals that nourish their young with milksecreted by mammary glands, preferably warm blooded mammals.

Immunogenicity: A measure of the ability of a targeting protein ortherapeutic moiety to elicit an immune response (humoral or cellular)when administered to a recipient. The present invention is concernedwith the immunogenicity of humanized antibody CC49.

Reduced immunogenicity: This refers to an antibody, typically humanized,that exhibits reduced immunogenicity relative to the parent antibody.

Immunoreactivity: A measure of the ability of an immunoglobulin torecognize and bind to a specific antigen.

Substantially similar binding properties: This refers to a humanizedantibody which retains the ability to specifically bind the antigenrecognized by the parent antibody used to produce the humanizedantibody. Preferably, the affinity of the humanized antibody is at leastabout 10% of the affinity of the parent antibody, more preferably atleast about 25%, even more preferably at least about 50%. Mostpreferably, the humanized antibody exhibits antigen-binding affinitythat is at least about 75% of the affinity of the parent antibody.Methods for assaying antigen-binding affinity are well known in the artand include half-maximal binding assays, competition assays, andScatchard analysis.

Substantially Homologous: Refers to immunoglobulin sequences thatexhibit at least about 85% identity, more preferably about 90% identity,most preferably about 95% identity with a reference immunoglobulin,wherein % identity is determined by comparing the number identical ofamino acid residues between the two immunoglobulins, wherein thepositions of the amino acid residues are indicated using the Kabatnumbering scheme.

Nomenclature: Nucleic acids, amino acids, peptides, protective groups,active groups and so on, when abbreviated, are abbreviated according tothe IUPAC IUB (Commission on Biological Nomenclature) or the practice inthe fields concerned.

DETAILED DESCRIPTION

To facilitate understanding of the invention, a discussion of thestructure of a typical antibody molecule will first be provided. Thebasic immunological structural unit is shown in FIG. 1. Antibodies (alsoreferred to as immunoglobulins) are constructed from four polypeptidechains, two heavy chains and two light chains. The two heavy chains arelinked to each other by disulfide bonds and each heavy chain is linkedto a light chain by a disulfide bond. There are two types of lightchain, lambda (Σ) and kappa (κ). There are five main heavy chain classes(or isotypes) which determine the functional activity of an antibodymolecule: IgM, IgD, IgG, IgA and IgE.

Each chain contains distinct sequence domains. The light chain includestwo domains, a variable domain (V_(L)) and a constant domain (C_(L)).The heavy chain includes four domains, a variable domain (V_(H)) andthree constant domains (C_(H)1, C_(H)2 and C_(H)3, collectively referredto as CH) The variable regions of both light (V_(L)) and heavy (V_(H))chains determine binding recognition and specificity to the antigen. Theconstant region domains of the light (C_(L)) and heavy (C_(H)) chainsconfer important biological properties such as antibody chainassociation, secretion, transplacental mobility, complement binding, andbinding to Fc receptors. The Fv fragment is the N-terminal part of theFab fragment of an immunoglobulin consisting of the variable portions ofone light chain and one heavy chain. The specificity of the antibodyresides in the structural complementarity between the antibody combiningsite and the antigenic determinant. Antibody combining sites are made upof residues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariableor framework regions (FR) influence the overall domain structure andhence the combining site.

The variants of the invention are derived from a humanized CC49(referred to as “parental HuCC49”). Parental HuCC49 is formed bygrafting all six (three heavy chain and three light chain) MAb CC49hypervariable regions onto the variable light (V_(L)) and variable heavy(V_(H)) frameworks of the human MAbs LEN and 21/28′CL, respectively,while retaining murine framework residues that may be required for theintegrity of the antigen combining site structure (FIG. 11). (Kashmiriet al., (1995) Hybridoma, 14:461-473). The variants of the inventioncontain a reduced murine content, and consequently, reducedimmunogenicity, when compared to HuCC49. Nonetheless, the variants ofthe invention retain a binding affinity that is substantially similar tothat of HuCC49. Preferably the binding affinity is at least about 10⁸M⁻¹. As used herein, HuCC49 refers to the humanized antibody formed byKashmiri et al. The terms “variant HuCC49” or “variant” refer to theimmunoglobulins of the invention.

A first aspect of the invention provides CDR variants of humanizedmonoclonal antibody (HuCC49) in which less than all six (three heavychain and three light chain) Complementarity Determining Regions (CDRs)of CC49 are present. A second aspect of the invention provides SDRvariants of humanized monoclonal antibody (HuCC49) in which onlySpecificity Determining Regions (SDRs) of at least one CDR from CC49 arepresent.

CDR Variants

According to the invention, CDR variants are formed by replacing atleast one CDR of CC49 in HuCC49 with a corresponding CDR from a humanantibody. Preferably, the L-CDR1 or L-CDR2, or both, from CC49 arereplaced by a corresponding CDR from a human antibody. The inventorshave found that a variant in which any of L-CDR3, H-CDR1, H-CDR2 orH-CDR3 of CC49 are replaced by a corresponding CDR from a human antibodydo not retain significant binding affinity.

Binding Affinity of CDR Variants

According to the invention, CDR variants in which L-CDR1 or L-CDR2 ofCC49, or both, are replaced by a corresponding CDR from a human antibodyretain biological activity that is substantially similar to the bindingaffinity of the parental CC49. Generally, the CDR variants of theinvention have a binding affinity that is about 25% to about 50% if thebinding affinity of the parental CC49, more preferably about 50% toabout 75%, most preferably, about 75% to about 100%.

CDR variants in which H-CDR2 is replaced by a corresponding CDR from ahuman antibody that is only slightly immunoreactive with TAG-72. Inparticular, such variants have a relative binding affinity that is about300 fold less than that of CC49.

CDR variants in which L-CDR3, H-CDR1, or H-CDR3 are replaced by acorresponding CDR from a human antibody do not appear to retain anybinding affinity for TAG-72.

Immunogenicity of CDR Variants

The CDR variants that have a reduced immunogenicity when compared toHuCC49 formed by grafting all six (three heavy chain and three lightchain) CDR from CC49 onto the variable light (V_(L)) and variable heavy(V_(H)) frameworks of the human MAbs LEN and 21/28′CL, respectively.That is, the CDR variants of the invention are less likely to elicit ananti-idiotypic or HAMA response. Immunogenicity can be characterizedusing competition radioimmunoassays known in the art in which an“anti-CC49” antibody that recognizes the parental CC49 is exposed toboth the parental MAb and the variant. Generally, a reduction inimmunogenicity is reflected by a reduction in binding of the variant bythe anti-CC49 antibody.

CDR variants in which L-CDR1 or L-CDR2, or both, of CC49 are replaced bya corresponding CDR from a human antibody show a slight reduction inimmunogenicity, that is, the variants do not bind to the anti-CC49antibody as well as HuCC49.

CDR variants in which L-CDR3 or H-CDR2 of CC49, is replaced by acorresponding CDR from a human antibody show a substantial reduction inimmunogenicity. However, the inventors have found that such variantsalso show a substantial reduction in immunoreactivity.

CDR variants in which H-CDR1 or H-CDR3 or CC49 are replaced by acorresponding CDR from a human antibody do not show any measurablechange in immunogenicity.

SDR Variants

The inventors have discovered that all six CDR of CC49 need not bepresent in their entirely for the humanized antibody to retain activity.Only residues that are directly involved in antigen contact, theSpecificity Determining Residues (SDRs), are needed. SDR variants areformed by replacing at least one SDR of CC49 in HuCC49 with a residue ata corresponding position from a human antibody.

It should be noted that not all CDRs include SDRs. For example, it wasdetermined that L-CDR1 and L-CDR2 of CC49 do not have any SDRs.Therefore, in one variant of the invention, L-CDR1 and L-CDR2 arereplaced entirely with human CDRs. However, SDR variants can be formedby replacing residues within these CDRS with a corresponding humanresidue. L-CDR1 from CC49 and LEN differ at three positions, 27b, 27fand 29. Because residues 27b, 27f, 29 are not important for the bindingaffinity of CC49, a suitable SDR variant can include a correspondinghuman residue at any of these position, or at any combination of thesepositions. L-CDR2 from CC49 and LEN differ at position 53 only. Residue53 is not considered important for the binding affinity of CC49. Thus, asuitable variant can include a corresponding human residue at position53.

L-CDR3 of CC49 differs from LEN at three positions, 94, 96 and 97. Thepartially buried residue at position 97 is not important for the antigenbinding activity of CC49. Thus, a suitable SDR variant can include acorresponding human residue at position 97. However, positions 94 and 96of L-CDR3 are involved in ligand contact and should not be replaced togenerate a functional SDR variant.

H-CDR1 of CC49 and 21/28′CL differ at three positions, 31, 32 and 34.However, SDR variants which include a corresponding human residue atpositions 32 and 34 demonstrate no antigen binding affinity. Thus, afunctional SDR variant should not include a corresponding human residueat either of these positions.

H-CDR2 of CC49 differs from human MAb 21/28′CL at eleven positions. Theresidues at positions 60, 61, 62 and 64 are not required for antigenbinding activity. Therefore, a SDR variant of the invention can includea corresponding human residue at any of positions 60, 61, 62 and 64, orany combinations thereof Generally, H-CDR3 does not need to beconsidered when designing an SDR variant, because it does not show anyreactivity to patients' sera.

In a preferred embodiment, the variant includes a combination of CDRand/or SDR substitutions to generate a variant having reducedimmunogenicity and a binding affinity that is substantially similar tothat of parental CC49. Suitable combinations include CDR variants inwhich both L-CDR1 and L-CDR2 of CC49 are replaced by a corresponding CDRfrom a human antibody. Other suitable variants include a combination ofSDR and CDR substitutions. For example, a suitable variant can includecorresponding human residues at position 97 of the light chain inaddition to a substitution of L-CDR1 and/or L-CDR2 from CC49 with thecorresponding CDRs from a human antibody. In another preferredembodiment, the variant includes a substitution at position 97 on thelight chain in combination with substitutions at positions 60, 61, 62and 64 on the heavy chain. In yet another embodiment, the variantincludes a substitution at position 97 on the light chain in addition toa substitution of L-CDR1 and/or L-CDR2 from CC49 with the correspondingCDRs from a human antibody in combination with substitutions atpositions 60, 61, 62 and 64 on the heavy chain.

In addition to variants specifically described herein, other“substantially homologous” modified immunoglobulins can be readilydesigned and manufactured using various recombinant DNA techniques wellknown to those skilled in the art. For example, the framework regionscan be varied at the primary structure level. Moreover, a variety ofdifferent human framework regions may be used singly or in combinationas a basis for the variant. In general, modifications of the genes maybe readily accomplished by a variety of well-known techniques, such assite-directed mutagenesis.

Alternatively, polypeptide fragments comprising only a portion of theprimary antibody structure may be produced wherein the fragmentsubstantially retains the immunoreactive properties of the variant.These polypeptide fragments include fragments produced by proteolyticcleavage of intact antibodies by methods well known in the art, orfragments produced by inserting stop codons at the desired locationsnucleotide sequence using site-directed mutagenesis. For example, a stopcodon can be inserted after C_(H)1 to produce Fab fragments or after thehinge region to produce F(ab′)₂ fragments. Single chain antibodies andfusion proteins which includes at least an immunoreactive fragment ofthe variant are also included within the scope of the invention. Forexample, the variants may be directly or indirectly attached to effectormoieties having therapeutic activity. Suitable effector moieties includecytokines, cytotoxins, radionuclides, drugs, immunomodulators,therapeutic enzymes, anti-proliferative agents, etc. Methods forattaching antibodies to such effectors are well known in the art.

Binding Affinity of SDR Variants

L-CDR1 from CC49 and LEN differ at three positions, 27b, 27f and 29.Since L-CDR1 of CC49 can be replaced with the corresponding CDR from LENwithout any significant loss of antigen binding reactivity, residues27b, 27f, 29 are not considered important for the binding affinity ofCC49. Thus, a variant of the invention can include a corresponding humanresidue at any of these three positions, or any combination thereof, andretain a binding affinity that is substantially similar to that of theparent HuCC49.

In L-CDR2, CC49 and LEN differ at position 53 only. Since L-CDR2 of CC49can be replaced with the corresponding CDR from LEN without anysignificant loss of antigen binding reactivity, residue 53 is notconsidered important for the binding affinity of CC49. Thus, thehumanized antibody of the invention can include a corresponding humanresidue at residue 53 and retain a binding affinity that issubstantially similar to that of the parent HuCC49.

L-CDR3 of CC49 differs from LEN at three positions, 94, 96 and 97. Thepartially buried residue at position 97 is not important for the antigenbinding activity of CC49. Thus, the humanized antibody of the inventioncan include a corresponding human residue at position 97 and retain arelative binding affinity that is substantially similar to that of CC49.However, positions 94 and 96 of L-CDR3 appear to be involved in ligandcontact. Therefore, an SDR variant which includes a corresponding humanresidue at either position 94 or 96, or both will generally suffer totalor near total loss of antigen binding reactivity.

H-CDR1 of CC49 and 21/28′CL differ at three positions, 31, 32 and 34.SDR variants which include a corresponding human residue at positions 32and 34 demonstrate no antigen binding affinity.

H-CDR2 of CC49 differs from human MAb 21/28′CL at eleven positions. Theresidues at positions 60, 61, 62 and 64 do not appear to be required forantigen binding activity. Therefore the humanized antibody of theinvention can include a corresponding human residue at any of positions60, 61, 62 and 64, or any combinations thereof, and the variant willretain a binding affinity that is substantially similar to that of CC49.

Immunogenicity of SDR Variants

SDR variants are particularly beneficial because some CDRs that areimportant for immunoreactivity are also immunogenic (e.g., L-CDR3 andH-CDR2). Thus, the immunogenicity of various SDR replacements withinL-CDR3 and H-CDR2 were examined.

As shown in FIG. 2, L-CDR3 consists of residues 89-97 and H-CDR2consists of residues 50-65. The inventors have found that SDR variantswhich include a corresponding human residue in positions 32 and 34(found within H-CDR1) or at position 97 (found within L-CDR3) are stillimmunogenic. Whereas, SDR variants which include a corresponding humanresidue in positions 60, 61, 62, and 64 (found within H-CDR2) or atposition 94 (found within L-CDR3) show a reduction in immunogenicity.SDR variants which include a corresponding human residue in position 96(found within L-CDR3) do not appear to be immunogenic.

Generally, the residues found in H-CDR3 does not need to be consideredwhen designing SDR variants, because it does not show any reactivity topatients' sera.

Human Antibodies

Suitable human antibodies include, but are not limited to: ROY, AU, REI,HAU, HK101′CL, SCW, WEA, HK137′CL, HK134′CL, DAUDI′CL, WALKER′CL,GAL(1), LAY, WES, Vb′CL, HK102′CL, EU, DEN, AMYLOID BAN, MEV, Vd′CL,Va′CL, KUE, Ve′CL, V13′CL, V18A′CL, V19A′CL, V19B′CL, V18C′CL, NIM, CUM,GM603CL, FR, RP M1-6410′CL, TI, WOL, SIE, NG9′CL, NEU, GOT, PAY, SON.GAR′, PIE, FLO, GLO, CUR, IARC/BL41′CL, POM, REE, K-EV15′CL, VJI′CL,VKAPPAIV, GERMLINE′CL, PB171′CL, LEN, NEWM, HA, NIG-64, NEW, BL2′CL,WAH, NIG-77, VOR, RHE, LOC,OKA, COX, NIG-51, NIG-84, MES, WH, NEI, WEIR,TOG, TRO, BOH, NIG-58, VIL, WIN, 41′CL, HIL, LAP, GAR, MOT, BO, MDG,AMYLOID-AR, SUT, THO,LBV′CL, NIG-48, HG3′CL, ND′CL, COR, DAW, OU, MCE′,CE-1′CL, HE, SUP-T1, VH-JA′CL, HIG1′CL, TUR, LAMDA-VH26′CL, WAS, H11′CL,TEI, BRO′IGM, GRA′, ZAP, JON, DOB, NIE, 333′CL, IHI′CL, IBIi′CL, 126′CL,112′CL, 115′CL, KOL and 21/28′CL. New human antibodies are beingdiscovered and sequenced, many of those, as of yet unknown antibodiesmay also be suitable. Preferably, human antibody has a sequence that isidentical or substantially similar (containing as few mutations aspossible) to the human germ line sequences. For example, the light chainCDR of CC49 in HuCC49 can be replaced with the corresponding CDR fromLEN (Kabat et al., 1991) and the heavy chain CDR can be replaced withthe corresponding CDR from 21/28′CL (Kabat et al., 1991).

Methods of Producing

The variants of the invention can be produced by expressing theappropriate DNA sequence in a host after the sequence has been operablylinked to (i.e., positioned to ensure the functioning of) an expressioncontrol sequence. Such expression vectors are typically replicable in ahost organism either as episomes or as an integral part of the hostchromosomal DNA. The expression vectors typically contain expressioncontrol sequences compatible with the host cell, such as an origin ofreplication. In addition, the expression vector will typically include apromoter. Suitable promoters include the polyhedrin promoter, lactosepromoter system, a tryptophan promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. Promoters typicallycontrol expression of the gene, optionally, with operator sequences, andhave ribosome binding site sequences and the like for initiating andcompleting transcription and translation. Commonly, expression vectorswill contain selection markers. DNA sequences encoding the light chainand heavy chain of the antibody may be inserted into separate expressionvectors, or into the same expression vector.

Suitable hosts include prokaryotic strains such as E. coli; Bacilli,including Bacillus subtilus; enterobacteriacae, including Salmonella,Serratia and Psuedomonas. Suitable hosts also include eukaryotic hostssuch as yeast, including Saccharomyces; Pichia pastoris; Sf9 insectcells; Sp2/0, VERO and HeLa cells, Chinese hamster ovary (CHO) celllines; W138, BHK, COS-7 and MDCK cell lines.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, which vary depending on thetype of cellular host. For example, calcium chloride transfection,calcium phosphate treatment, electroporation or cationic liposomemediated transfection (such as DOTAP). Successfully transformed cells,can be identified by a variety of techniques well known in the art fordetecting the binding of a receptor to a ligand.

Once expressed, the gene products can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, and gel electrophoresis.Substantially pure immunoglobulins of at least about 90% to about 95%homogeneity are preferred, and 98% to 99% or more homogeneity mostpreferred for pharmaceutical uses.

Methods of Use

Once purified, the variants of the invention may be usedtherapeutically, or in developing and performing assays, in vivo or invitro diagnostic procedures, and imaging. The variants of the inventionare particularly useful for the treatment of diseases such as cancer, inparticular for treating or detecting cancer. The variants can beadministered to a patient alone or in combination with a pharmaceuticalformulation. Typically, the variants are incorporated into apharmaceutically acceptable, non-toxic, sterile carrier as a suspensionor solution. The antibodies of the invention can be used as separatelyadministered compositions or given in conjunction with chemotherapeuticor immunosuppressive agents.

The variants provide unique benefits when used for the treatment ofcancer. In addition to the ability to bind specifically to malignantcells and localize tumors without binding to non-cancerous cells, thevariants have a reduced immunogenicity when compared to HuCC49.

For diagnostic purposes, the antibodies may either be labeled orunlabeled. Unlabeled antibodies can be used in combination with otherlabeled antibodies (second antibodies) that are reactive with thehumanized antibody, such as antibodies specific for human immunoglobulinconstant regions. Alternatively, the antibodies can be directly labeled.A wide variety of labels can be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands(particularly haptens), etc. Numerous types of immunoassays areavailable and are well known to those of skill in the art.

Kits according to the present invention include frozen or lyophilizedvariant to be reconstituted by thawing or by suspension in a liquidvehicle. The kits may also include a carrier or buffer. Preferably, thekit also comprises instructions for reconstituting and using the variantantibody.

WORKING EXAMPLES

To identify the CDRs essential for binding, a panel of variant HuCC49MAbs were generated using the baculovirus expression system. HuCC49 wasprepared by grafting MAb CC49 CDRs onto the V_(L) and V_(H) frameworksof the human MAbs LEN and 21/28′ CL, respectively, as described byKashmiri et al., (1995) Hybridoma, 14:461-473. Six CDR variants wereconstructed by replacing a single CC49 CDR of either the light or heavychain with the corresponding human antibody CDR (LEN and 21/28′CL,respectively). Variants were denoted as L-1, L-2, L-3, H-1, H-2 or H-3.A seventh variant, L-1,2 was made by replacing two CC49 light chain CDRs(L-CDR1 and L-CDR2) with the corresponding CDRs of the human antibodyLEN.

Since the seven CDR variants were derived by simply replacing the murineCDRs with the human antibody hypervariable regions, all of the variantscarry identical V_(H) and V_(L) frameworks and K1 and k chain constantregions SDR heavy chain and light variants were constructed bysubstituting mutagenic nucleotides in or near the CDRs.

Example I Preparation of CDR Substituted MAb CC49

According to the invention, CDR variants are formed by replacing atleast one CDR of CC49 in HuCC49 with a corresponding CDR from a humanantibody. The CDR variants of the invention include:

-   -   Variant L-1: L-CDR1 of CC49 (SEQ ID NO: 1) was replaced with        L-CDR1 of LEN (SEQ ID NO: 7).    -   Variant L-2: L-CDR2 of CC49 (SEQ ID NO: 2) was replaced with        L-CDR2 of LEN (SEQ ID NO: 8).    -   Variant L-3: L-CDR3 of CC49 (SEQ ID NO: 3) was replaced with        L-CDR3 of LEN (SEQ ID NO: 9).    -   Variant L-1,2: L-CDR1 and L-CDR2 of CC49 (SEQ ID NOs: 1 and 2,        respectively) were replaced with L-CDR1 and L-CDR2 of LEN (SEQ        ID NOs: 7 and 8, respectively).    -   Variant H-1: H-CDR1 of CC49 (SEQ ID NO: 4) was replaced with        H-CDR1 of 21/28′CL (SEQ ID NO: 10).    -   Variant H-2: H-CDR2 of CC49 (SEQ ID NO: 5) was replaced with        H-CDR2 of 21/28′CL (SEQ ID NO: 11).    -   Variant H-3: H-CDR3 of CC49 (SEQ ID NO: 6) was replaced with        H-CDR3 of 21/28′CL (SEQ ID NO: 12).

Production of Oligomers to Generate V_(H) Variants

Synthesis of three variant V_(H) genes was performed using the overlapextension PCR technique described by Kashmiri et al., (1995) Hybridoma14:461-473. Four 123-126 base pair long overlapping oligonucleotides(SEQ ID NOs: 15-18), (which together encompass the entire sequence ofthe variant V_(H) gene on alternating strands) were used to generatevariant V_(H) genes. (FIG. 12B) The oligomers were supplied by MidlandCertified Reagent Co., Midland, Tex. Instead of a template DNA, the PCRmixture contained 2 pmoles of the four oligonucleotides. PCR was carriedout by three cycles of a denaturing step at 94° C. for 1 minute, aprimer annealing step at 55° C. for 2 minutes, and an extension step at70° C. for 2 minutes, followed by 17 additional cycles of denaturation(94° C., 1 minute), primer annealing (55° C., 2 minutes), and extension(72° C., 1 minute). All polymerase chain reactions (PCRs) were carriedout in a final volume of 100 Tl of PCR buffer containing 100 TM ofdNTPs, 5 units of Taq DNA polymerase (Boehringer Mannheim) and 20 pmolof each end primer.

Production of Oligomers to Generate V_(L) Variants

The three variant V_(L) genes were generated using 30-43 baseoligonucleotides as a mutagenic primer. The oligonucleotides containedthe desired base changes in the targeted CDR. The mutagenic primers forthe V_(L) genes were synthesized using a Model 8700 DNA synthesizer(Miligen/Bioresearch, Burlington, Vt.). (FIG. 12A) Primer inducedmutagenesis was carried out by a two-step PCR method, as described byLandt et al., (1990) Gene, 96:125-128. pLNXCHuCC49HuK (Kashmiri et al,(1995) Hybridoma 14:461-473) (FIG. 2) was used as a template in bothsteps. In the first step, the mutagenic primer was used as a 3′ primerwhile a 20 nucleotide long end primer served as a 5′ primer. The productof the first PCR was gel purified and utilized as a 5′ primer for thesecond PCR in which a 20 nucleotide long end primer was used as a 3′primer. The 20 nucleotide long end primers used for DNA amplificationwere supplied by Midland Certified Reagent Co. (Midland, Tex.). Thesequences for these primers are reported by Kashmiri et al., (1995)Hybridoma 14:461-473 and are as follows: (SEQ ID NO: 23) 1. 5′ V_(H),5′-CTA AGC TTC CAC CAT GGA G-3′ (SEQ ID NO: 24) 2. 3′ V_(H), 5′-ATGGGC CCG TAG TTT GGC G-3′ (SEQ ID NO: 25) 3. 5′ V_(L), 5′-GCA AGC TTC CACCAT GGA TA-3′ (SEQ ID NO: 26) 4. 3′ V_(L), 5′-AGC CGC GGC CCG TTT CAGTT-3′Each of the primers carries a single restriction endonuclease site atits flank. The 5′ primers carry a HindIII site, while the 3′ V_(H)primer carries an ApaI, and the 3′ V_(L) primer has a SacII site. Therestriction endonuclease recognition sequences are underlined.

The first PCR was carried out in a final volume of 100 Tl containing 10ng of the template DNA, 20 pmol each of the 3′ and 5′ primers, 100 TMdNTPs and 5 units of Taq DNA polymerase (Boehringer Mannheim,Indianapolis, Ind.). Each step of the PCR consisted of 25 cycles ofdenaturation (94° C., 1 minute), primer annealing (45° C., 2 minutes),and extension (72° C., 2 minutes). The PCR product was extracted withphenol/chloroform, precipitated with ethanol and gel purified prior toinsertion into a vector.

Example II Assembly of CDR Substituted MAb CC49 PCR Products

The PCR products encoding the V_(H) were treated with HindIII/ApaI. ThePCR products were subcloned for sequencing in pBluescript S/K+(Stratagene, La Jolla, Calif.) at a HindIII/ApaI site after the plasmidwas linearized using the appropriate restriction endonucleases. Insertswere sequenced to check their fidelity to their templates.

To assemble the variable and constant regions of the heavy chain theHindIII/ApaI insert was released form pBluescript. A DNA fragmentencoding the human K1 constant region was excised from pLgpCXHuCC49HuG1(Kashmiri et al, (1995) Hybridoma 14:461-473), (FIG. 3) by ApaI/ClaIcleavage. The HindIII/ApaI and the ApaI/ClaI fragments were joined. Therecombinant was unidirectionally inserted, by three way ligation,between the HindIII and ClaI sites of pBluescript. The DNA sequenceencoding the entire heavy chain was then cleaved from pBluescript byHindIII/ClaI digestion. Its termini were filled in using the Klenowfragment of the DNA polymerase. The insert was subcloned in a lightchain construct of pAcUW51 (FIG. 4), at the blunt ended BamHI sitelocated downstream of the polyhedrin promoter.

The PCR products encoding the V_(L) were treated with HindIII/SacII. ThePCR products were subcloned for sequencing in pBluescript S/K+(Stratagene, La Jolla, Calif.) at a HindIII/SacII site after the plasmidwas linearized using the appropriate restriction endonucleases. Insertswere sequenced to check their fidelity to their templates.

To assemble the variable and constant region of the light chain, theHindIII/SacII insert was released from the pBluescript construct. A DNAfragment encoding the human kappa constant region was excised frompLNCXHuCC49HuK (Kashmiri et al, (1995) Hybridoma 14:461-473), (FIG. 2)by SacII/ClaI treatment. The HindIII/SacI fragments were joined to theHindIII/ClaI linearized pBluescript by three way ligation. The entirelight chain was cleaved from pBluescript using EcoRI. The EcoRI fragmentwas inserted into the baculovirus expression vector pAcUW51 (Pharmingen,San Diego, Calif.) at the EcoRI site located downstream from the p10promoter.

The baculovirus expression construct of the parental HuCC49 wasgenerated using DNA fragments encoding HuCC49 heavy and light chainsobtained from PLNCXHuCC49HuK and pLgpCXHuCC49HuG1. PLNCXHuCC49HuK wascleaved with HindIII. The resulting >1.0 Kb DNA fragment encodingHuCC49Huk was subcloned in pBluescript at the HindIII site. Theresulting construct was then cleaved with BamHI and the fragment wascloned in the baculovirus vector pAcUW51 at the BamHI site, downstreamfrom the polyhedrin promoter. A >1.4 Kb DNA fragment encoding HuCC49HuG1was cloned from pLgpCXHuCC49HuG1 using HindIII/ClaI. The DNA fragmentwas filled using the Klenow fragment of DNA polymerase. pAcUW51 waslinearized with BglII and its ends blunted using the Klenow fragment.The DNA fragment was then inserted in the pAcUW51 expression constructof HuCC49HuK, downstream from the p10 promoter.

Example III Generation of Baculovirus Recombinant CDR Substituted CC49MAb

Serum free adapted Sf9 insect cells (Gibco BRL, Gaithersburg, Md.) werecultured at 28° C. in Sf900-II medium (Gibco BRL) without supplements asdescribed by Salgaller et al, (1993) Cancer Res., 53:2154-2161. Todevelop the recombinant baculovirus, 1×10⁶ Sf9 cells in a 35 mm dishwere co-transfected with 0.5 ml pAcUW51 derived baculovirus expressionconstruct of the CDR substituted light chain gene and the HuCC49 heavychain gene along with linearized BACULOGOLD wild type baculovirus DNA(Pharmingen), using a cationic liposome mediated transfection system,DOTAP (Boehringer Mannheim) according to the suggested protocol.Similarly, variant antibodies containing CDR substituted heavy chainwere produced by co-transfecting Sf9 cells with BACULOGOLD baculovirusDNA and baculovirus dual expression constructs carrying CDR substitutedheavy chain and HuCC49 light chain genes. Baculovirus recombinant HuCC49(hereafter referred to as HuCC49) was used as a control antibody. HuCC49was produced by transfecting insect cells with pAcUW51 carrying HuCC49light and heavy chains.

Five days after transfection, the infectious supernatants were harvestedfrom the transfectants. 1 ml of this supernatant was serially dilutedand used to infect a monolayer of 5×10⁶ Sf9 cells in a 100 mm dish. Thecells were then overlaid with 0.5% Baculovirus Agarose (Invitrogen,Carlsbad, Calif.) as described by Bei et al., (1995) J. Immunol.Methods, 186:245-255. Viral plaques were expanded by three rounds ofinfection. For each round of expansion, a larger population of freshlyseeded monolayers of Sf9 cells were infected, using the highestproducing clone as a source of inoculum. The putative recombinant viralplaques were purified and isolated in 1 ml of Sf900 media. If necessary,viruses were further amplified by infecting cells at an Multiplicity ofInfection (MOI) of 0.1. To produce the recombinant antibodies, 6.0×10⁸Sf9 cells were infected with the infectious supernatant at an MOI of 5.

Purification of CDR Substituted MAbs

The culture supernatant was clarified by pelleting cell debris at10,000×g, and was applied to an ion-exchange column (DE52; Whatman,Hillsboro, Oreg.) at pH 7.2 to remove extraneous proteins. The unboundprotein fraction was subjected to protein G (Gibco BRL) affinitychromatography. The material bound to protein G was eluted from thecolumn using 0.1 M glycine hydrochloride buffer, pH 2.6 and the pH ofthe eluted material was immediately adjusted to 7.4 using 1.0 M Trisbuffer, pH 8.0. The buffer was replaced by phosphate buffered saline andthe eluted material was concentrated using a Centricon 30 microconcentrator (Amicon, Beverly, Mass.). Protein concentration wasdetermined by the method of Lowry et al., (1951) J. Biol. Chem.193:265-275. The purity of the antibody preparation was analyzed using aprecast continuous 4-15% SDS-polyacrylamide Tris-glycine gel (NovexSystems, San Diego, Calif.) and visualized by Coomassie blue staining asdescribed by Kashmiri et al., (1995) Hybridoma 14:461-473.

Radiolabeling of MAbs

The murine MAb CC49 and HuCC49 were labeled with Na ¹²⁵I using theiodogen (Pierce, Rockford, Ill.) method as described by Fraker (1978)Biochem. Biophys. Res. Commun., 80:849-857 and Colcher (1988) CancerRes., 48:4597-4603. The protocol routinely resulted in specificactivities at 5-10 TCi/Tg. The immunoreactivities of the radiolabeledMAbs were assessed by the radioimmunoassay described by Schott et al.,(1992) Cancer Res., 52:6413-6417 using bovine submaxillary mucin (BSM)immobilized on a solid support (Reacti-gel HW 65F; Pierce)

Immunoglobulin Production

The titer of the transfectants and the putative viral plaques wereassayed for immunoglobulin production by enzyme-linked immunosorbentassay (ELISA) based on reactivity of the test aliquot with goatanti-human Fc (K1) and goat anti-human kappa antibodies as described byBei et al., (1995) J. Immunol. Methods, 186:245-255. Transfectants andviral plaques derived from each of the expression constructs werepositive for immunoglobulin production.

However, when the transfectants and the viral plaques were assayed forimmunoreactivity with TAG-72 positive bovine submaxillary mucin (BSM),the clones derived from the expression constructs carrying L-1, L-2 andL-1,2 were positive, while those generated by the H-2 expressionconstruct were barely immunoreactive. Those derived from the constructscarrying either L-3, H-I and H-3 demonstrated no immunoreactivity withBSM at all.

It was then assessed whether the poor or lack of BSM reactivity of theclones derived from L-3, H-1, H-2 and H-3 expression constructs was dueto low levels of immunoglobulin secretion by these clones. To that end,Sf9 cells were infected with the infectious supernatants at an MOI of 5and cultured under the conditions described above. The secreted antibodywas purified from equal volumes of the culture supernatant from each ofthe infected cultures, and analyzed by SDS-PAGE. The gel profile undernon-reducing conditions showed that the mobility of the variantantibodies was identical to that of the HuCC49, which has a molecularweight of approximately 160 kDa (data not shown). Under reducingconditions, the variant antibodies, like the HuCC49 MAb, yielded twoprotein bands of approximately 25-28 kDa and 50-55 kDa (FIG. 6). Thesemobilities are in conformity with the molecular masses of theimmunoglobulin heavy and light chains. More importantly, it is evidentthat regardless of their BSM reactivity, clones derived from each of theconstructs encoding CDR-substituted heavy or light chain produce as muchimmunoglobulin as the clone derived from the constructs encoding theparental humanized heavy and light chains.

Example IV Competition Radioimmunoassays for CDR Substituted Variants

Binding Affinity of Variant Antibodies

The relative binding affinity of the HuCC49 and the CDR substitutedvariant antibodies to TAG-72 was determined using the competitionradioimmunoassay (RIA) described by Milenic et al., (1991) Cancer Res.,51:6363-6371. Serial dilutions of the purified variant MAbs, as well asthe parental HuCC49, were prepared in phosphate buffered saline (PBS)containing 1% bovine serum albumin (BSA). 25 Tl was added to the wellsof microtiter plates containing 10 ng BSM. ¹²⁵I-labeled HuCC49 (50,000cpm in 25 Tl) was then added to each well. The plates were incubatedovernight at 4° C. and then washed and counted in a K-scintillationcounter.

Unlabeled HuCC49 or its variants were used to compete for the binding of¹²⁵I-HuCC49 to TAG-72 positive BSM. The variants, L-1, L-2 and L-1,2,were found to completely inhibit the binding of the ¹²⁵I-labeled HuCC49to TAG-72, while L-3 did not compete at all (FIG. 7).

The relative affinity constants were calculated by the modification ofthe Scatchard method described by Frankel et al., (1979) Mol. Immunol.,16: 101-106. An approximation of the specific activity of the¹²⁵I-HuCC49 was made and used to determine the final concentration foreach of the dilutions of the variant MAbs. The calculations wereperformed as described by Milenic et al., (1991) Cancer Res.,51:6363-6371.

The relative affinity constants (Ka) of the variants were as follows:

-   -   L-1 had a Ka of 3.3×10⁻⁸ M (only about 2-fold less than that of        HuCC49).    -   L-2 had a Ka of 6.81×10⁻⁸ M (comparable to that of HuCC49).    -   L-1,2 had a Ka of 2.9×10⁻⁸ M (only about 2-fold less than that        of HuCC49).    -   H-1 and H-3 displayed no competition    -   H-2 competed only slightly with the HuCC49. The Ka of H-2 was        0.018×10⁻⁸ M (approximately 300-fold less than the Ka of        HuCC49).

Reactivity of the CC49 Anti-Idiotypic Antibodies to the VariantAntibodies

The variant MAbs were also characterized in the competitionradioimmunoassay RIA described by Irvine et al., (1993) Cancer Immunol.Immunother., 36:281-292 using mouse anti-idiotypic MAb generated againstMAb CC49. Three anti-idiotypic (AI49-8, AI49-3 and AI49-1) wereselected, representing each of the anti-idiotypic subsets, I, Θ, and K,respectively. In the same manner described above, 100 ng of MAb AI49-3(Θ-subset), AI49-1 (K-subset) or AI49-8 (I-subset) were adsorbed to eachwell of a 96-well microtiter plate. 25 Tl of the serially dilutedvariant MAbs or HuCC49 was added to each well along with 25 Tl of¹²⁵I-murine CC49. The plates were washed and counted after an overnightincubation at 4° C.

The results for the light chain variants are shown in FIG. 8. For theAI49-3 (Θ-subset): L-CDR1 appears to be only partially involved in therecognition of CC49 by AI49-3; L-CDR2 does not appear to be involved inthe recognition of CC49 by AI49-3; and L-CDR3 appears to be importantfor recognition of CC49 by AI49-3. For the AI49-1 (K-subset): L-CDR1appears to be not required for recognition of CC49 by AI49-1; L-CDR2appear to be only modestly involved in the recognition of CC49 byAI49-1; and L-CDR3 appears to be important for recognition of CC49 byAI49-1. For the AI49-8 (I-subset): neither L-CDR1, L-CDR2, nor L-CDR3appear to have any influence on the interaction of AI49-8 with CC49.

The results for the heavy chain variants are shown in FIG. 9. For theAI49-3 (Θ-subset): H-CDR1 and H-CDR3 do not appear to be involved inbinding of HuCC49 to AI49-3, while H-CDR2 appears to be important forrecognition of CC49 by AI49-3 (approximately 4-15 times more competitoris required for 50% inhibition by H-2 as compared to HuCC49). For theAI49-1 (K-subset): H-CDR1 and H-CDR3 do not appear to be involved inbinding of HuCC49 to AI49-1, while H-CDR2 appears to be important forrecognition of CC49 by AI49-1 (approximately 4-15 times more competitoris required for 50% inhibition by H-2 as compared to HuCC49). For theAI49-8 (I-subset): H-CDR1 and H-CDR3 do not appear to be involved inbinding of HuCC49 to AI49-8, while H-CDR2 appears to be important forrecognition of CC49 by AI49-8 (there is a complete loss of inhibition bythe variant).

An analysis of patient reactivity to the variants of HuCC49 show thatthree of the 6 CDRs (L-CDR2, H-CDR1 and H-CDR3) do not seem to berecognized by the patient, while L-CDR1 and H-CDR2 appear to be involvedin the patient's recognition of HuCC49 to some degree. L-CDR3 (which isimportant for antigen binding) is the immunodominant CDR recognized bythe patient. L-CDR3 is immunodominant in mice as well (AI49-1 andAI49-3, the two anti-idiotypic antibodies that inhibit antigen bindingof HuCC49, require L-CDR3 for recognition of HuCC49).

Example V High Performance Liquid Chromatography

The CDR variants were further characterized using the serum from apatient that had received ¹⁷⁷Lu-CC49 in a phase 1 radioimmunotherapyclinical trial (Mulligan et al., (1995) Clin. Cancer Res., 1: 1447-1454.Several of the patients in this study were found to have anti-idiotypicantibodies to MAb CC49. One patient was selected to perform apreliminary study to identify whether any of the CC49 CDRs wereimmunodominant.

Using a modification of the method reported by Colcher et al., (1990) J.Nucl. Med., 31:1133-1142 and Mulligan et al., (1995) Clin. Cancer Res.,1: 1447-1454, serial dilutions of the purified CDR variants wereincubated with the patient's sera along with ¹²⁵I-labeled HuCC49.Specifically, the method of Colcher and Mulligan was modified asfollows: prior to the study, HAMA and TAG-72 were removed from the seraby adsorption with CC92 conjugated solid support. The amount of serarequired for half maximal complex formation with HuCC49 was thendetermined. Specifically, 8 Tl of patient sera was mixed with >500,000cpm of ¹²⁵I-HuCC49 and serial dilutions of purified HuCC49 or itsvariants. The preparations were brought to a final volume of 50 Tl.

The ability of the variants to inhibit complex formation of the patentsera with ¹²⁵I-labeled HuCC49 was monitored using HPLC analysis. 25 Tlof each solution was applied to a TSK3000 analytical column (7.8 mm×30cm; Tosohaas, Montgomeryville, Pa.) and eluted at 0.5 ml/min with 100 mMKCl in 67 mM sodium phosphate (pH 6.8). Radioactivity was monitoredusing a flow-through K-scintillation detector (Model 170, Beckman).

If the variant contained the CDR recognized by the patient, then thevariant would compete with the radiolabeled HuCC49 and complex formationwould not occur and there would not be an alteration in the retentiontime of the ¹²⁵I-HuCC49. If the variant no longer contained a CDRrecognized by the patient, then complex formation would result. Thus,the ability of the CDR variants to inhibit complex formation of thepatient sera with the radiolabeled HuCC49 was determined by theretention time of the ¹²⁵I-HuCC49. The percent inhibition of complexformation was calculated and plotted versus concentration of eachcompetitor to evaluate the degree of the patient's reactivity with theindividual CDR variants. FIG. 15 shows a comparison of patientreactivity with HuCC49 and CDR variants.

-   -   L-1 (variant without CC49 L-CDR1) showed some inability to        inhibit complex formation. Thus L-CDR1 appears to be somewhat        involved in immunogenicity (0.7 Tg of competitor was required        for 50% inhibition of complex formation).    -   L-2 appeared to compete better than parental HuCC49 by 2 fold        (an enhanced recognition by the patient)    -   L-3 showed no inhibition of complex formation, thus L-CDR3        appears necessary for immunogenicity    -   L-1,2 demonstrated some inability to inhibit complex formation,        indicating that L-CDR1 and/or L-CDR2 are somewhat involved in        immunogenicity.    -   H-1 inhibits complex formation and therefore contributes to        immunogenicity.    -   H-2 showed little complex formation, thus H-CDR2 does not appear        to be necessary for immunogenicity (10 Tg of competitor was        unable to achieve 50% inhibition of complex formation).    -   H-3 demonstrated some inability to inhibit complex formation,        thus H-CDR3 appears to be somewhat involved in immunogenicity        (0.4 Tg of competitor was required for 50% inhibition of complex        formation).

Example VII Preparation of SDR Substituted MAb CC49

Padlan et al., (1995) FASEB J., 9:133-139 disclose that the SDRs oflight chain are bounded by positions 27d and 34; 50 and 55; and 89 and96. The heavy chain SDRs are contained within positions 31 and 35b; 50and 58; and 95 and 101.

FIG. 2 shows the differences between the amino acid residues of thelight chain CDRs of CC49 and LEN, and the heavy chain CDRs of CC49 and21/28′CL.

In L-CDR1, CC49 and LEN differ in three residues; at positions 27b, 27fand 29. The residues at positions 27b (a buried residue) and 27f werefound not to be directly involved in ligand contact, while the one atposition 29 was found to interact with ligand in two complexes; in oneonly by main chain atoms. Residue 27b is located outside the suggestedSDR boundaries. Residues 27f and 29 are well within the suggested SDRboundaries.

In L-CDR2, CC49 and LEN differ at position 53 only, and this positionwas found to be involved in ligand contact in only three of the 31complexes of known structure. Residue 53 is well within the suggestedSDR boundaries.

Since L-CDR1 and 2 of CC49 were replaced with their counterparts fromLEN without any significant loss of antigen binding reactivity (above),it was concluded that residues 27b, 27f, 29 and 53 were not importantfor binding of CC49 to its antigen. L-CDR1 and L-CDR2 of CC49 were notconsidered for the mutation experiments because they were replaced withthe corresponding CDRs of the human MAb LEN without significant loss ofantigen binding reactivity.

The immunodominant L-CDR3 of CC49 differs from LEN at three positions,94, 96 and 97. Each of the three residues of CC49 L-CDR3 was replacedwith the residue present at the corresponding position in the LEN CDR togenerate light chain variants ⁹⁴L, ⁹⁶L and ⁹⁷L, respectively. Anotherlight chain variant, ^(94,97)L was generated carrying two substitutions,one at position 94 and the other at 97. Two additional variants werederived from the HuCC49 light chain variant L_(1,2), in which the L-CDR1and L-CDR2 of CC49 were earlier replaced with their counterparts fromthe human MAb LEN. One variant, ⁹⁷L_(1,2), carried a single substitutionat position 97. The other, ^(94,97)L_(1,2), had substitutions at twopositions, 94 and 97.

Of the three residues that differ between L-CDR3 of CC49 and LEN, apartially buried residue at position 97 was not important for theantigen binding activity of CC49. This residue is not located within thesuggested boundary of SDRs of the L-CDR3. Thus, variant ⁹⁷L did not showany loss in antigen binding activity. Variant ⁹⁷L_(1,2) showed only aninsignificant loss of antigen binding activity.

Positions 94 and 96 of L-CDR3 are involved in ligand contact in 19 and22 known antibody:antigen complexes, respectively. Thus it wasconsistent that variants ⁹⁶L and ⁹⁴L suffered total and near total lossof antigen binding reactivity. When the mutation at position 94 wasimposed on the variants ⁹⁷L and ⁹⁷L_(1,2), it destroyed their antigenbinding function.

H-CDR1 of CC49 and 21/28′CL differ at three positions, 31, 32 and 34.The residue at position 31 is directly involved in ligand binding in 12of the 31 complexes; in five of those, only main chain atoms wereinvolved. The residue at position 32 is ligand contacting in eight ofthe 31 complexes of known structure. The residue at position 34 isinvolved in ligand contact in none of the 31 complexes of knownstructure. Residues at positions 32 and 34 of the CC49 H-CDR1 werereplaced with the corresponding residues of 21/28′CL MAb (^(32,34)H) totest whether position 32 is important for ligand contact and ineliciting anti-idiotypic response.

H-CDR2 of CC49 differs from human MAb 21/28′CL at eleven positions. Theresidues at positions 60, 61, 62 and 64 were not ligand contacting inany of the complexes of known structure. Therefore, these residues ofCC49 were prime candidates for replacement. Accordingly, a heavy chainvariant of HuCC49, ^(60-62,64)H, was generated by replacing theseresidues of HuCC49 with their counterparts in human MAb 21/28′CL.

H-CDR3 was not considered for mutations, because it did not show anyreactivity to patient's sera (above).

The following SDR variants were made:

-   -   Variant ⁹⁴L: residue 94 of CC49 L-CDR3 was replaced with the        residue present at the corresponding position in LEN.    -   Variant ⁹⁶L: residue 96 of CC49 L-CDR3 was replaced with the        residue present at the corresponding position in LEN.    -   Variant ⁹⁷L: residue 97 of CC49 L-CDR3 was replaced with the        residue present at the corresponding position in LEN.    -   Variant ^(94,97)L: residue 94 and 97 of CC49 L-CDR3 was replaced        with the residue present at the corresponding position in LEN.    -   Variant ⁹⁷L_(1,2): derived from the HuCC49 light chain variant        L_(1,2), in which the L-CDR1 and L-CDR2 of CC49 were replaced        with their counterparts from the human MAb LEN; residue 97 of        CC49 L-CDR3 was replaced with the residue present at the        corresponding position in LEN.    -   Variant ^(94,97)L_(1,2): derived from the HuCC49 light chain        variant L_(1,2), in which the L-CDR1 and L-CDR2 of CC49 were        replaced with their counterparts from the human MAb LEN;        residues 94 and 97 of CC49 L-CDR3 were replaced with the residue        present at the corresponding position in LEN.    -   Variant ^(32,34)H: residues at positions 32 and 34 of the CC49        H-CDR1 were replaced with the corresponding residues of 21/28′CL        MAb.    -   Variant ^(60-62,64)H: residues at positions 60, 61, 62 and 64 of        the CC49 H-CDR2 were replaced with the corresponding residues of        21/28′CL MAb.

Production of Oligomers

The oligomers were produced essentially as described in Example 1.pLgpCXHuCC49HuK1, the expression construct for parental HuCC49 heavychain genes was used as the template for heavy (^(32,34)H and^(60-62,64)H) chain variant gene synthesis. pLNCXHuCC49HuK, theexpression construct of the parental HuCC49 light chain gene was used asa template for the light (⁹⁴L, ⁹⁶L, ⁹⁷L and ^(94,97)L) chain variantgene synthesis. Variants L₁ and L_(1,2) were developed by replacing onlythe L-CDR1 or both L-CDR1 and L-CDR2 of CC49, respectively, with theirLEN counterparts. For the synthesis of ⁹⁴L_(1,2) and ^(94,97)L_(1,2)genes, an expression construct of the L_(1,2) variant in a baculoviralexpression construct was used as a template.

Mutagenic oligonucleotide primers, ranging in size from 37 to 56nucleotides, were synthesized using a Model 8700 DNA synthesizer(Milligen/Bioresearch, Burlington, Vt.). They were purified on oligo-Pakcolumns (Milligen/Bioresearch) according to the supplier'srecommendation. The sequences of the mutagenic primers were as follows,where the mutagenic changes are underlined: V_(L) CDR3: (SEQ ID NO: 27)5′-GCC AGC GCC GAA GCT GAG GGG ATA GCT ATA ATA CTG CTG ACA-3′ (SEQ IDNO: 28) 5′-GGT GCC AGC GCC GAA GCT GAG GGG GGT GCT ATA ATA CTG CTGACA-3′ (SEQ ID NO: 29) 5′-GCC ACG GCC GAA TGT GTA GGG ATA GCT ATA ATACTG CTG ACA-3′ (SEQ ID NO: 30) 5′-GCC GAA TGT GAG GGG GGT GCT ATA ATACTG CTG ACA ATA-3′ VH CDR1: (SEQ ID NO: 31) 5′-GTT TCA CCC AGTGCA TTG CAT AAT CAG TGA AGG TGT A-3′ VH CDR2: (SEQ ID NO: 32) 5′-GTG GCCTTG CCC TGG AAC TTC TGT GAG TAC TTA AAA TCA TCG TTT CCG GGA GAG AA-3′

Example VIII Assembly of PCR Products

The PCR products were assembled and sequenced as described in ExampleII. The 425 base pair (bp) PCR product obtained using the HuCC49 lightchain construct as a template carried sequences encoding the leaderpeptide, the CC49 V_(L) domain and the amino terminus of the kappa (κ)constant region, terminating in a SacII site located 10 bp downstream ofthe V_(L). Similarly, the 432 base pair (bp) PCR product from the heavychain template encompassed sequences encoding the leader, the V_(H) andthe amino terminus of the C_(H)1 domain, extending to the ApaI site,which is located 17 bp downstream from the start of the C_(H)1 domain.

Generation of Recombinant SDR Substituted CC49 MAb

SDR substituted variants were generated essentially as described ExampleIII, except for the following. The Sf900-II medium included 50 Tg/ml ofantibiotic, gentamicin and the infectious supernatants were harvestedsix days after transfection.

Purification of SDR Substituted CC49 MAb

Three days after infection, the tissue culture supernatant was harvestedand clarified by centrifugation at 2000×g for 10 minutes. Tris bufferwas added to the supernatant to a final concentration of 20 mM.Following incubation at 4° C. for 2-3 hours, any contaminating proteinswere pelleted by centrifugation at 10,000×g for 15 minutes. Thesupernatant was applied to a protein G agarose column (Gibco BRL) andthe bound protein was eluted from the column, using 0.1 M glycinehydrochloride, pH 2.5. The pH of the eluted material was immediatelyadjusted to 7.0 with 1.0 M Tris buffer, pH 8.0. The protein wasconcentrated using a Centriplus 30 microconcentrator (Amicon, Beverly,Mass.), centrifuged at 3000×g for 80 minutes. The concentrated proteinwas recovered in phosphate-buffered saline (PBS). The proteinconcentration was determined by the as described in Example III. Thepurity of the antibody preparation was evaluated by electrophoresis on4-12% SDS-PAGE, under reducing and non-reducing conditions. The proteinswere visualized by staining with Coomassie blue, as described in ExampleIII.

Example IX Competition Radioimmunoassays for SDR Substituted Variants

ELISA

The ability of the variants to express immunoglobulin molecules andtheir antigen reactivity of the heavy (^(32,34)H and ^(60-62,64)H) orvariant light (⁹⁴ L, ⁹⁶L, ⁹⁷L, ^(94,97)L, ^(Υ)L_(1,2) and^(94,97)L_(1,2) ) chain variants was evaluated using ELISA assays. ELISAassays were carried out by coating individual wells of a 96-wellpolyvinyl microtiter plates with 1 Tg/well of TAG-72 positive bovinesubmaxillary mucin (BSM) (Sigma Chem. Co., St. Louis, Mo.), andfollowing the procedure described by Bei et al., (1995) J. Immunol.Methods, 186:245-255.

Not all variant antibodies were positive for antigen binding activity.Results of the ELISA assay for the binding activity to the TAG-72positive BSM showed that the variant antibodies specified by expressionconstructs carrying the variant genes ³² ³⁴H and ⁹⁶L were not reactivewith BSM. In contrast, variant antibodies expressed by ⁹⁷L and^(60-62,64)H constructs showed strong BSM binding activity. Whileimmunoglobulin molecules expressed by ⁹⁴L and ⁹⁴L_(1,2) constructsshowed moderate positive antigen binding reactivity, those expressed by^(94,97)L_(1,2) were only weakly positive. (FIG. 13)

A partial or complete loss of antigen binding activity of the variantimmunoglobulins might be attributed to the detrimental effect of the SDRsubstitutions on the combining site of HuCC49. Alternatively, theplaques may show lower or no antigen binding reactivity because some ofthe expression constructs failed to express, were expressing atsignificantly lower level, or producing antibodies that were notphysically normal. To examine these possibilities, variant antibodieswere produced and purified from a larger batch of cells that werefreshly infected with inoculum derived from the highest producing clonefor each of the constructs. The concentration of the secreted variantantibodies in culture supernatants ranged between 2-3 Tg/ml. Purifiedimmunoglobulin molecules were characterized by SDS-PAGE. Under reducingconditions, immunoglobulin molecules expressed by each of the constructsyielded two bands that co-migrated with the heavy and light chains ofHuCC49 MAb (data not shown) Antibodies produced by the insect cellsharboring expression constructs ⁹⁷L_(1,2) and ^(94,97)L_(1,2) genespaired with the HuCC49 heavy chain gene showed similar results (data notshown). These results make it evident that all constructs expressed andproduced comparable levels of immunoglobulin molecules of appropriatesize. Therefore, it can safely be concluded that the variant HuCC49 MAbscarrying ⁹⁶L and ^(32,34)H substitutions suffered a total loss ofantigen binding activity.

Competition Radioimmunoassay

Competition radioimmunoassays (RIAs) were performed to determinerelative binding of the variant MAbs and the parental HuCC49 to BSM.Details of the procedure are described by Kashmiri et al., (1995)Hybridoma, 14:461-473. Serial dilutions of the purified unlabeledvariant antibodies or the parental HuCC49 MAb were used to compete withradiolabeled HuCC49 for binding to the TAG-72 positive BSM. Briefly, 25Tl of serial dilutions of the purified SDR substituted variants or theparental HuCC49 in PBS containing 1% BSA were added to wells of 96-wellmicrotiter plates containing 10 ng of BSM. 25 Tl of ¹²⁵I-labeled HuCC49(50,000 cpm) was added to each well to compete with the unlabeledparental or variant HuCC49 for binding to the BSM coated on the plates.The plates were incubated overnight at 4° C. and then washed and countedin a K-scintillation counter.

Competition profiles of the light chain variants presented in panel Ashow that the variant ⁹⁶L failed to compete, while all other variantsantibodies competed with the parental HuCC49 completely and with similarslopes. (FIG. 13) However, the competition curves of all variants withthe exception of ⁹⁷L were shifted significantly to the right, indicatinga loss of reactivity with antigen (BSM). This shift was notably lesspronounced for ⁹⁷L_(1,2). Similarly, it is evident from the competitionprofiles of the heavy chain variants (panel B) that the variant MAb^(32,34)H, with substitutions in H-CDR1, did not inhibit binding ofHuCC49 MAb to BSM, whereas ^(60-62,64)H, the variant with substitutionsin the H-CDR2, competed completely with a profile that was almostidentical to that of the parental HuCC49.

The relative affinity constants were calculated as described in ExampleIV. The relative affinity constants (Ka) of the variants were calculatedfrom the linear parts of the competition curves. The Ka of ⁹⁷L and^(60-62,64)H MAbs were 3.6×10⁸ M⁻¹ and 2.2×10⁸ M⁻¹, respectively. Thesevalues are comparable to 3.2×10⁸ M⁻¹, the Ka of the parental HuCC49. Thevariant ⁹⁷L_(1,2) was found to have a Ka of 1.4×10⁸ M⁻¹, which isapproximately 2- to 3-fold less than the Ka of HuCC49 MAb.

Two new expression constructs were then generated and expressed in Sf9cells; in one of them, the gene encoding the variant heavy chain^(60-62,64)H was paired with the gene encoding the light chain variant⁹⁷L. Gene ^(60-62,64)H was paired with the ⁹⁷L_(1,2) light chain gene inthe other construct. Competition profiles of the purified antibodiesshow that these variant MAbs competed completely with HuCC49 MAb forantigen binding, yielding competition curves of the same slope asHuCC49. (FIG. 13) The relative affinity constant of the Variant MAb⁹⁷L/^(60-62,64)H was 5.48×10⁸ M⁻¹, a figure favorably comparable to thatof HuCC49, while the Ka of the variant MAb ⁹⁷L_(1,2)/^(60-62,64)H was1.15×10 M⁻¹, which is about 3-fold less than that of the parental HuCC49MAb.

Example X High Performance Liquid Chromatography

In a reported Phase I clinical trial, in which ¹⁷⁷Lu-labeled MAb CC49was administered to adenocarcinoma patients, several patients were foundto have anti-idiotypic antibodies to MAb CC49. Sera collected from thestudy was used to examine the potential immunogenicity of the variants.The sera was obtained by separating the blood by centrifugation. HighPerformance Liquid Chromatography (HPLC) was used to determine antigenreactivity of the variants by monitoring complex formation betweenantibodies in the patient sera and the variant MAbs.

Prior to HPLC analysis, any free TAG-72 and human anti-murine antibodiesother than anti-idiotypic antibodies to CC49 present in the sera wereabsorbed out using MAb CC92 conjugated to a solid support. MAb CC92 is amurine anti-TAG-72 antibody which as the same isotype as CC49 andrecognizes an epitope of TAG-72 other than that recognized by CC49.Patient sera was then incubated with ¹²⁵I-labeled HuCC49 (approximately500,000 cpm) and 5 Tg of the cold competitor; either HuCC49 or one ofthe variant MAbs.

The competition assay is described in Example V. Briefly, patient serawas mixed with >0.3 TCi of ¹²⁵I-HuCC49 and serial dilutions of purifiedHuCC49 or its variants. Prior to the assay, the amount of sera requiredin half-maximal immune complex formation was determined. The mixture wasbrought to a final volume of 50 Tl. 25 Tl of the final solution wasapplied to a 7.8 mm×30 cm TSK3000 analytical column (Tosohaas,Montgomeryville, Pa.) and eluted at 0.5 ml/min with elution buffer (100mM KCl in 67 mM sodium phosphate, pH 6.8). Radioactivity was monitoredusing a flow-through Model 170 K-scintillation detector (Beckman).

Complex formation of the radiolabeled HuCC49 with the anti-idiotypicantibodies in patient sera reduced the retention time of the radiolabelon the column. The ability of the variant to inhibit complex formationwith ¹²⁵I-labeled HuCC49 was determined by the differential in theretention time of the radiolabel on HPLC column, when a mixture of seraand ¹²⁵I-labeled HuCC49 was loaded on the column with or withoutincubation with the cold competitor. Inhibition of complex formation bya competitor indicates that the competitor shares the immunogenicepitope with HuCC49. (FIG. 14)

From an analysis of the percent of input counts recovered as a complex,when a mixture of ¹²⁵I-labeled HuCC49 and sera from each of the fourpatients was incubated with 5 Tg of cold competitor and subjected toHPLC analysis, it is evident that the variant antibodies ⁹⁷L and^(32,34)H, like HuCC49, inhibited complex formation. In contrast, thevariant MAbs ⁹⁶L and ^(94,97)L_(1,2), like the nonspecific Humanimmunoglobulin did not inhibit complex formation of HuCC49 with serafrom any patient except EA. Complex formation with EA sera was partiallyinhibited by the two variants. The variant MAbs ⁹⁴L, ^(94,97)L,⁹⁷L_(1,2) and ^(60-62,64)H inhibited complex formation only partiallywith sera from all patients. The variant ⁹⁷L/^(60-62,64)H, whose antigenbinding activity was comparable to that of parental HuCC49, inhibitedsera of three patients (DG, CP and DS) only partially, but completelyinhibited the sera from EA patient to form complexes with HuCC49. Moreimportantly, the variant ⁹⁷L_(1,2)/^(60-62,64)H did not compete withHuCC49 to form complex with anti-idiotypic antibodies present in serafrom two patients (CP and DS) while showing only partial competitionwith sera from two other patients (DG and DS).

Using serial dilutions of the competitors, competition profiles weredeveloped to determine the relative amounts of unlabeled competitorantibodies required to achieve 50% competition of the binding of¹²⁵I-labeled HuCC49 to the anti-idiotypic antibodies present in serafrom one of the patients (CP). The percent inhibition of complexformation was calculated and plotted versus the concentration ofcompetitor.

The competition profiles show that the cold HuCC49 competed completelyand it required approximately 250 ng of the parental HuCC49 antibody toachieve 50% competition. In contrast, variant ⁹⁷L_(1,2)/^(60-62,64)Hinhibited binding of the radiolabeled HuCC49 to the sera anti-idiotypicantibodies only minimally; even 1 Tg of the variant failed to achievemore than 25% competition, that was achieved by 60 ng of HuCC49. Thisvariant, which retains moderate antigen binding activity and reacts withpatient's sera only minimally, might be most advantageous for clinicalapplications. This variant was further studied for plasma clearance andbiodistribution in an animal model.

FIG. 16 is a graph showing the immunoreactivity of variant⁹⁷L_(1,2)/^(60-62,64)H to human sera containing anti-murine CC49variable region antibodies as assessed by HPLC analysis. The percentinhibition of the complex formation was calculated and plotted versus ngof the competitors. The competitors were HuCC49 (▪) and variant (□).

Example XI Biodistribution and Pharmacokinetic Studies

Pharmacokinetics

Since the rate of plasma clearance has a bearing on in vivo tumortargeting, a comparison of the pharmacokinetics of the variant to theparental HuCC49 was assessed using the procedures described by Kashmiriet al., (1995) Hybridoma, 14:461-473.

To study pharmacokinetics, athymic mice bearing TAG-72 positive LS-174Ttumors (Colcher et al., (1983) Cancer Res., 43:736-742) were injectedintravenously in the tail vein with a mixture containing 1.4 TCi¹³¹I-labeled HuCC49 and 4.4 TCi ¹²⁵I-labeled variant MAb⁹⁷L_(1,2)/^(60-62,64)H. Blood samples were collected at various timepoints via the tail vein into 10 Tl heparinized capillary tubes(Drummond, Broomall, Pa.). The amounts of ¹³¹I and ¹²⁵I in the plasmawere determined and corrected for the respective rates of the decay ofthe two radionuclides. The percentage of the injected dose of eachradionuclide remaining in the plasma was then calculated for each timepoint. The results suggest that the blood clearance patterns of the twoantibodies are not significantly different. (FIG. 17). For 50% of theinjected dose of the HuCC49 or variant to clear the blood compartment,required 1 and 2 hours, respectively. At 24 hours, 85% and 80% of theradiolabeled HuCC49 and the variant, respectively, was cleared from theblood. At 48 hours, the percentage of HuCC49 and the variant clearedfrom the blood was 92% and 88%, respectively.

Biodistribution

Biodistribution assays were performed as described by Kashmiri et al.,(1995) Hybridoma, 14:461-473. To investigate the ability of the variantHuCC49 MAb to localize to human tumor xenograft and determineradiolocalization index (RI), athymic mice bearing TAG-72 positiveLS-174T tumors (Colcher et al., (1983) Cancer Res., 43:736-742) wereinjected intravenously in the tail vein with a mixture containing 1.4TCi ¹³¹I-labeled HuCC49 and 4.4 TCi ¹²⁵I-labeled variant MAb⁹⁷L_(1,2)/^(60-62,64)H. The amount of ¹³¹I and ¹²⁵I were determined inblood samples collected via tail vein at specified times. For each timepoint, 5 mice were sacrificed to collect and weigh tumor, blood and allother major organs. Radioactivity was measured in a K-scintillationcounter and it was corrected for the decay. The percentage of theinjected dose per gram (% ID/gm) for each organ was determined.

The % injected dose of the two antibodies per gram of either tumor ordifferent normal tissues that were collected at different time pointsshows that the biodistribution patterns of the two antibodies areessentially the same. Both showed significant tumor localization by 24hours. (FIG. 18) By 48 hours, when only 8% and 12% of the injected dosewas present in the blood, 17.6% and 23.8% ID/b of HuCC49 and the variantwere, respectively, present in the tumor.

Example XII Characterization of Humoral Immune Response against CC49

In this Example, the humoral immune response against HuCC49CDR-replacement variants is examined.

Generation of Humanized CC49 (HuCC49) and Humanized CC49 CDR-ReplacementVariants (CDR Variants)

A clone producing humanized CC49 (HuCC49) was grown in protein freehybridoma medium PFHM-II (GIBCO-BRL, Gaithersburg, Md.) as described byKashmiri (1995), Hybridoma, 14:461-473. The humanized CC49 monoclonalantibody (MAb) was purified from the tissue culture supernatant byProtein G affinity chromatography as described by Kashmiri (1995),Hybridoma, 14:461-473.

Seven HuCC49 CDR-variants were produced as described in Examples I-III.

Radiolabeling

MAb HuCC49, BL-3 and the CDR-replacement variants of HuCC49 were labeledwith Na ¹²⁵I, using the iodogen method (Pierce, Rockford, Ill.) asdescribed by Fraker et al. (1978), Biochem. Biophys. Res. Commun.80:849-857; and Colcher et al. (1988), Cancer Res., 48:4597-4603. BL-3is an isotype-matched control for CC49 (described by Colcher et al.(1987), Cancer Res., 47:4218-4224). The labeling procedure typicallyresulted in specific activities of 5-10 TCi/Tg.

Patients and Sample Collection

Patients with recurrent metastatic adenocarcinoma were enrolled in aPhase I Study to assess the maximum tolerated does of intravenouslyadministered ¹⁷⁷Lutetium radiolabeled MAb CC49 (Mulligan, (1995) Clin.Cancer Res. 1: 1447-1454).

In the Phase I Study, adenocarcinoma patients were given a test dose of0.1 mg (i.v. bolus) of MAb CC49 and observed for 30 minutes prior toadministration of the ¹⁷⁷Lu-labeled MAb CC49. The radiolabeled MAb wasgiven as a 1 hour i.v. infusion. Blood samples were collected prior toand at the end of the infusion, and 0.5, 1 and 2 hours after theinfusion, and afterward, daily for 7 days. Patients returned for afollow-up examination 10 at 3, 6 or 8 weeks, at which time blood sampleswere collected. Sera was separated and stored at −20° C. until analyzed.Sera from these patients provided a resource for assessing the humoralresponse of patients to the murine MAb CC49. The patient characteristicsare presented in Table 1, below. TABLE 1 Patient CharacteristicsDose^(a) Dose Level Patient Age Sex Tumor mCi mg MAb 10 mCi/m² DS 52 FBreast 16.0 20 LW 45 F Breast 19.0 20 JJ 61 F Breast 17.2 20 25 mCi/m²DG 45 F Breast 41.0 20 LJ 45 F Breast 40.3 20 JM 42 F Breast 45.4 20 15mCi/m² JG 61 M Colon 29.8 44 RW 46 F Lung 24.2 20 TD 50 M Colon 31.5 47EA^(b) 53 F Colon 24.2 20 CP^(b) 53 F Colon 26.0 20 LQ^(b) 45 F Colon29.7 20^(a)Patients were administered ¹⁷⁷Lu-PA-DOTA-CC49 by intravenousinjection.^(b)Patient received new formulation of ¹⁷⁷Lu-PA-DOTA-CC49 that waslabeled using a modification of the method described by Mulligan et al.(1995), Clin. Cancer Res. 1: 1447-1454.

PA-DOTA was conjugated to human serum albumin (HSA), radiolabeled withNa ¹²⁵I, incubated with the patient sera and analyzed for immune complexformation by size-exclusion HPLC. None of the sera showed detectablereactivity with the PA-DOTA-HSA conjugate (Data not shown).

Determination of Patient Humoral Response

The sera from the twelve patients was evaluated for the presence ofhuman anti-murine antibodies (HAMA) in response to MAb CC49 using highperformance liquid chromatograph (HPLC) as described by Mulligan et al.(1996) Clin. Cancer Res., 1:1447-1454. The analysis was performed byadding about 500,000 cpm (0.4TCi) of ¹²⁵I-BL-3 to 50 Tl of patient sera.Following a 60 minute incubation at 37° C., 25 Tl of the mixture wasapplied to a size-exclusion column (TSK 3000SW; TosoHaas,Montgomeryville, Pa.) equilibrated in 67 mM sodium phosphate (pH 6.8)containing 100 mM KCl. The sera samples were eluted at a flow rate of0.5 ml/min. The protein was detected by absorbance at 280 nm and theradioactivity was measured using a flow-through K-scintillation counter(Model 170, Beckman Instruments, Inc., Berkeley, Calif.). The presenceof HAMA was indicated by a shift in the elution profile of the ¹²⁵I-BL-3because the formation of immune complexes with the radiolabeled BL-3results in a shorter retention time. The patients' pre-study sera,normal human sera and phosphate buffered saline with ¹²⁵I-BL-3 were usedas controls. A patient with a known HAMA response from a previous study(Colcher et al. (1990), J. Nucl. Med., 31:1133-1142) served as apositive control. The patients' sera were demonstrated to haveantibodies against the variable region of the murine CC49.

FIG. 19 shows an HPLC analysis of patient HAMA following intravenousinjection of ¹⁷⁷Lu-CC49. Serum samples from LQ were analyzed for thepresence of HAMA at various timepoints before and after injection with20 mg of ¹⁷⁷Lu-labeled CC49. Pre-study sera (A), sera collected at 7days (B), 3 weeks (C), and 6 weeks (D) were mixed with ¹²⁵I-BL-3 andapplied to a size exclusion column. Reduction in retention time of theradiolabeled BL-3 as compared to migration of the ¹²⁵I-BL-3 in buffer(E) were indicative of immune complex formation and therefore thepresence of HAMA.

Lack of complex formation is evident (FIG. 19A) when the pre-study seraof Patient LQ is incubated with the ¹²⁵I-BL-3. All of the radioactivityis associated with the peak at about 18.5 minutes, the same retentiontime for ¹²⁵I-BL-3 in buffer (FIG. 19E). Complex formation is alsoabsent when the sera collected at seven days is incubated with ¹²⁵I-BL-3(FIG. 19B). With sera collected at 3 weeks (FIG. 19C), however, there isan indication of complex formation (46%) with the appearance of twopeaks with a shorter retention time (i.e., 14 and 16 minutes). The peaksat a shorter retention time indicate the development of a highermolecular weight species in the sera. At 6 weeks (FIG. 19D), the HAMAresponse has increased, the amount of radioactivity bound in complexesis now 66%.

FIG. 20 shows an HPLC analysis of patients' humoral response to thevariable region of MAb CC49. The percent complex formation has beenplotted versus time for (solid lines) patients DS (◯), LW (□), JJ (Δ),DG (●), LJ (▪), TD(▴); (dotted lines) JG (◯), RW (□), JM (Δ), EA (●), CP(▪), LQ (▴);

At one week, none of the patients showed a detectable response againstthe HuCC49 (FIG. 20). At 3 weeks, sera from nine of the twelve patients(75%) appears to contain antibody against the variable region of CC49with one patient having a notably higher response than the others. Forthe eleven patients evaluated at six weeks, only two patients did notelicit a human antivariable region antibody response (HAVRA) againstCC49, i.e., 9 of 11 evaluable patients (82%) had antibody against thevariable region of the murine MAb CC49.

Three patterns of HAMA-HAVRA response are evident. The patterns of theHAMA and HAVRA responses elicited in each of the patients were verysimilar, differing only in the apparent level of antibody. Patients DG,LW, LQ and CP developed HAVRA simultaneously with HAMA. Patients DS andJM appear to have a strong HAVRA, while HAMA response is modest. Whilein patients TD, JG, and EA, the HAVRA level is lower than HAMA at 3weeks, followed by HAMA and HAVRA attaining high levels at latertimepoints. In no patient was there a HAVRA response without thedevelopment of HAMA.

The HAMA results for the twelve patients are summarized below in Table2. TABLE 2 HPLC Analysis of Patients' Anti-mouse immunoglobulin responseafter i.v. injection of ¹⁷⁷Lu-CC49 Days Post-Injection of ¹⁷⁷Lu-CC49Patient 7 21 42 56 DS 0^(a) 1 16  27 LW 3 6 81 NA JJ 0 12 3  4 DG 0 2484 NA LJ 0 42 NA NA JM 0 8 47 NA JG 4 83 83 NA RW 0 1 2 NA TD 0 95 100NA EA 0 27 100 100 CP3 0 33 27 NA LQ 0 46 66 100^(a)The values are the percent of ¹²⁵I-BL-3 detected in complexes aftera brief incubation with the patient sera and resolved by size-exclusionchromatography. The timepoints of each patient are background correctedusing the patients' pre-study sera.

The patterns of the HAMA responses are varied and are consistent withprevious findings by Colcher et al. (1990), J. Nucl. Med. 31:1133-1142.Ten out of the twelve patients (83%) demonstrate a HAMA response at 3weeks following a single intravenous injection of 20 mg ¹⁷⁷Lu-labeledCC49, two patients (LW and JG) have minimal responses evident at 7 dayswith complexes of 3% and 4%, respectively. One patient (RW) may beconsidered a nonresponder. Some of the patients show an escalating HAMAresponse, while others plateau. Yet another (JJ) peaks at 3 weeks,followed by an apparent decrease in the HAMA level. Overall, at 3 weeks,8 of 12 patients (57%) at and 6 weeks, 9 of 11 (82%) were HAMA positive.

Specificity of Patient Response

The specificity of the patients' antibody response to CC49 was assessedusing ¹²⁵I-labeled HuCC49 and HuCC49 CDR-replacement variants todetermine whether or not any of the responses were directed against thevariable region of CC49. To accomplish this, the HPLC methodology wasemployed using ¹²⁵I-HuCC49 as the probe (See, Kashmiri et al. (1995),Hybridoma, 14:461-473).

To eliminate the artifactual influence of TAG-72 in the HPLC analysisfor anti-CC49 antibody responses found in the patient's serum,immunoadsorbents were prepared as reported by Ferroni et al. (1992) J.Clin. Lab. Analysis, 4:465-473. For the purpose of these studies,purified MAb CC92 was coupled to Reacti-gel (HW65F, Pierce) according tothe method of Heam et al. (1979), J. Chromatog, 185:463-470. MAb CC92 isa second-generation monoclonal antibody that reacts with TAG-72, butwith an epitope distinct from the one recognized by CC49.

Before probing the patients' sera with the ¹²⁵I-HuCC49, removal of HAMAand circulating TAG-72 were confirmed using ¹²⁵I-BL-3 and ¹²⁵I-B72.3,respectively (data not shown). MAb B72.3 is an anti-TAG-72 MAb that hasbeen shown to form complexes with TAG-72 in patient sera (Colcher et al.(1990), J. Nucl. Med., 31:1133-1142).

In the competition assay, 5 Tg of the cold competitor (either purifiedHuCC49 or one of its variants) was added to a mixture of patient sera(collected 8 weeks post-i.v. injection with ¹⁷⁷Lu-CC49) and ¹²⁵I-HuCC49and then analyzed by size-exclusion chromatography for the absence orpresence of complexes. The percent inhibition of complex formation wascalculated. If the variant competed with the ¹²⁵I-labeled MAb, andcomplex formation was inhibited, then the variant still contained theimmunodominant CDR. If the variant failed to inhibit complex formation,then the CDR that is no longer present in the variant is recognized bythe patient and hence it is an immunogenic CDR. An example of this assay(using serum from patient LQ) is shown in FIG. 21. Panel A is theprofile of the HuCC49 in buffer only. Panel B, is the profile showingcomplex formation (42.9%) resulting from patient sera (LQ) incubatedwith ¹²⁵I-HuCC49. When HuCC49 is added as a competitor, there iscompetition for the ¹²⁵I-HuCC49 and a loss or absence of complexes isobserved (Panel C). The same is true of a variant which still containsan immunogenic CDR (e.g., light chain CDR2 as the competitor) (Panel D).In contrast, there is either a partial (Panel F) or total retention ofthe complexes (Panel E), when light chain CDR1 or CDR3 variants,respectively, are the competitors.

The results are very striking, see Table 3. TABLE 3 HPLC Analysis ofPatient Reactivity to CDR-Replacement variants of HuCC49^(a) CompetitorPatient CDR^(b) DS DG JG EA CP LQ None — 33.5^(c) 46.2 24.5 56.8 32.242.9 HuCC49 — 0 0 2.6 0.5 1.5 3.0 Hu IgG — 46.4 59.0 25.1 63.6 ND 54.1Light Chain 1 16.0 12.2 9.8 10.1 16.9 14.3 2 2.7 3.4 2.7 4.4 3.0 2.4 334.8 48.2 22.4 37.6 33.5 46.7 1, 2 24.6 24.5 12.6 19.4 15.7 20.2 HeavyChain 1 10.2 3.9 3.3 7.0 5.8 3.5 2 32.7 32.5 12.7 24.7 29.7 36.6 3 7.35.1 3.7 8.2 6.7 4.6^(a)The sera from patients injected with ¹⁷⁷Lu-CC49 were tested forreactivity with variants of HuCC49 in which individual CDRs had beensubstituted with human sequences in both the heavy and light chains ofHuCC49. Five Tg of the purified CDR-replacement variants were added to amixture of ¹²⁵I-HuCC49 and the patient sera and then analyzed for thepresence or absence of immune complex formation.^(b)The number indicates which CDR in the HuCC49 has been replaced witha human CDR sequence.^(c)The values are the percent of complexes, the higher molecular weightspecies, resolved by size-exclusion chromatography.

Of the six patients analyzed, all six demonstrated reactivity with CDR3light chain indicating that light chain CDR3 may be immunodominant inmurine CC49 MAb. In the heavy chain, CDR2 appears to be dominant but notwith the same level of consensus (four of the six patients show the samelevel of reactivity, the other two demonstrated partial reactivity).Concordance was obtained among the six patients in regard to CDR2 of thelight chain and CDR1 and CDR3 of the heavy chain, which do not appear tocontribute to the immunogenicity of the MAb. This is also the case withthe light chain CDR1 and, it follows, the variant with the dualsubstitution of CDR1 and 2 in the light chain, in which all six patientsdisplayed a partial recognition of the variants. Partial recognitionwith the heavy chain CDR2 variant with two patients may be due to a lossof part but not all of the cognizant epitope, a change in theconformation or conformational epitope, or loss of amino acid residuesthat might stabilize the antibody:antibody interaction.

Quantitation of Patient Antibody Response

Quantitation of the HAMA or anti-variable region antibody levels in fourpatients was performed using HPLC analysis. The quantitation study wasperformed by adding either 500 ng of unlabeled BL-3 or 250 ng of HuCC49,respectively, to the mixture of patient serum and ¹²⁵I-HuCC49 andcalculating the amount of BL-3 or HuCC49 bound in complexes.

As shown in Table 4, below, at 6 weeks, the amount of HAMA varies frompatient to patient by 43-fold, while the variability of HAVRA is within4-fold. Furthermore, the HAMA versus HAVRA levels may vary from 10 to145-fold. Clearly, HAVRA can be detected at 3 weeks, and, notsurprisingly, it does not appear to attain the same levels as HAMA. Inpatient EA, there is a dramatic 1 0-fold increase in the level of HAVRAfrom 6 to 8 weeks that is noteworthy. TABLE 4 Quantitation of anti-CC49variable region and anti-murine response of patients administered¹⁷⁷Lu-CC49 Tg of Ab/ml Sera Post-Mab Patient Injection BL-3^(a)HuCC49^(b) EA 0 0 0 3 weeks 4.1 0.3 6 weeks 289.0 2.3 8 weeks 314.4 21.6CP 0 0 0 3 weeks 16.0 0.8 5 weeks 25.2 0.7 6 weeks 23.2 0.7 LQ 0 0 ND 3weeks 4.61 0.4 6 weeks 6.64 0.7 8 weeks ND 1.7 JG 0 0 0 3 weeks 58.6 0.76 weeks 47.8 2.6

Competition Radioimmunoassay

To confirm whether the HAVRA was actually an anti-idiotypic response,including internal image anti-idiotypic antibodies, to the murine MAbCC49, the sera from one patient (EA) was selected and assessed forblocking of the binding of ¹²⁵I-HuCC49 to BSM in a radioimmunoassay.

The immunoreactivity of the radiolabeled MAbs was assessed using bovinesubmaxillary mucin (BSM) immobilized on a solid support (Reacti-GelHW65, Pierce) as a modification of the method reported by Heam et al.(1979), J. Chromatog., 185:463-470 and Schott (1992) Cancer Res.,52:6413-6417. Briefly, 15 bovine submaxillary mucin (BSM), which isTAG-72 positive, was adsorbed to each well of a 96-wellpolyvinylchloride microtiter plate at 10 ng in 50Tl of phosphatebuffered saline (pH 7.2) as described by Horan Hand et al. (1992),Cancer Immunol. Immunother., 353: 165-174. After treating the wells with5% BSA in PBS, serial dilutions of the patient sera (25 Tl in 1% BSA inPBS) were added to each; ¹²⁵I-CC49 (38 nCi in 25 Tl) was also added.Following an 18 hour incubation at 4° C., the plates were washed and thewells counted in a K-scintillation counter. The percent inhibition wascalculated and compared to that of unlabeled CC49. Human IgG (OrganonTeknika, Durham, N.C.), which does not react with TAG-72 was included asa control antibody.

It was found that the patient sera could block the binding of¹²⁵I-HuCC49 with BSM (FIG. 22) suggesting that the patient, inactuality, demonstrates an anti-idiotypic response, consisting of theinternal image anti-idiotypic antibodies. Furthermore, theanti-idiotypic response was observed to increase over an eight weekperiod. FIG. 22 shows the detection of patient (EA) anti-idiotypicantibody response to murine CC49: pre-study sera from patient EA({circumflex over (∘)}); sera collected at 3 weeks (A), 6 weeks (B), and8 weeks (C).

All references cited in this disclosure are hereby incorporated byreference.

1. A recombinant nucleic acid sequence encoding a humanized anti-TAG-72CC49 antibody, wherein the humanized anti-TAG-72 antibody comprises: alight chain Complementarity Determining Region (L-CDR)1, a L-CDR2, and aL-CDR3; and a heavy chain Complementarity Determining Region (H-CDR)1, aH-CDR2, and a H-CDR3, wherein L-CDR3, H-CDR1, H-CDR2 and H-CDR3 comprisemurine monoclonal CC49 antibody Complementarity Determining Regions(CDRs) and at least one of L-CDR1 and L-CDR2 are a human monoclonal LENantibody L-CDR1 and L-CDR2, and wherein the humanized CC49 antibodyretains binding affinity for TAG-72 and has reduced immunogenicity, ascompared to a parental humanized CC49 antibody.
 2. The recombinantnucleic acid sequence of claim 1, wherein L-CDR1 is the human monoclonalLEN antibody L-CDR1.
 3. The recombinant nucleic acid sequence of claim1, wherein L-CDR2 is the human monoclonal LEN antibody L-CDR2.
 4. Therecombinant nucleic acid sequence of claim 1, wherein both L-CDR1 andL-CDR2 are human monoclonal LEN antibody L-CDR1 and L-CDR2,respectively.
 5. The recombinant nucleic acid sequence of claim 1,wherein the parental humanized CC49 antibody comprises three L-CDRs andthree H-CDRs of the murine monoclonal CC49 antibody, a variable lightchain framework of a human monoclonal LEN antibody, and a variable heavychain framework of a human monoclonal 21/28′CL antibody.
 6. Arecombinant nucleic acid sequence comprising a promoter sequenceoperably linked to the recombinant nucleic acid sequence of claim
 1. 7.A recombinant vector comprising the recombinant nucleic acid sequence ofclaim
 6. 8. A host cell transformed with the recombinant vector of claim7.
 9. A recombinant nucleic acid sequence encoding a humanizedanti-TAG-72 CC49 antibody, wherein the humanized anti-TAG-72 antibodycomprises: a light chain Complementarity Determining Region (L-CDR)1, aL-CDR2, and a L-CDR3; and a heavy chain Complementarity DeterminingRegion (H-CDR)1, a H-CDR2, and a H-CDR3, wherein L-CDR3, H-CDR1, H-CDR2and H-CDR3 are of a murine CC49 monoclonal antibody and L-CDR1 andL-CDR2 are of a human monoclonal LEN antibody L-CDR1 and L-CDR2respectively, and wherein at least one amino acid at position 60, 61,62, or 64 in the murine CC49 H-CDR2 is replaced with an amino acid at acorresponding position in the human monoclonal 21/28′CL antibody,wherein the humanized CC49 antibody retains binding affinity for TAG-72and has reduced immunogenicity, when compared to a parental humanizedCC49 antibody.
 10. The recombinant nucleic acid sequence of claim 9,wherein an asparagine at position 60 in the murine CC49 H-CDR2 isreplaced with a serine, a glutamic acid at position 61 in the murineCC49 H-CDR2 is replaced with a glutamine, an arginine at position 62 inthe murine CC49 H-CDR2 is replaced with a lysine, a lysine at position64 in the murine CC49 H-CDR2 is replaced with a glutamine, or athreonine at position 97 of the murine CC49 L-CDR3 is replaced with aserine.
 11. The recombinant nucleic acid sequence of claim 9, whereinthe parental humanized CC49 antibody comprises three L-CDRs and threeH-CDRs of the murine monoclonal CC49 antibody, a variable light chainframework of a human monoclonal LEN antibody, and a variable heavy chainframework of a human monoclonal 21/28′CL antibody.
 12. A recombinantnucleic acid sequence comprising a promoter sequence operably linked tothe recombinant nucleic acid sequence of claim
 9. 13. A recombinantvector comprising the recombinant nucleic acid sequence of claim
 12. 14.A host cell transformed with the recombinant vector of claim
 13. 15. Arecombinant nucleic acid sequence encoding a humanized anti-TAG-72 CC49antibody, wherein the humanized anti-TAG-72 antibody comprises: a lightchain Complementarity Determining Region (L-CDR)1, a L-CDR2, and aL-CDR3; and a heavy chain Complementarity Determining Region (H-CDR)1, aH-CDR2, and a H-CDR3, wherein L-CDR3, H-CDR1, H-CDR2 and H-CDR3 are of amurine CC49 monoclonal antibody and L-CDR1 and L-CDR2 are of a humanmonoclonal LEN antibody L-CDR1 and L-CDR2 respectively, and wherein athreonine at position 97 in the murine CC49 L-CDR3 is replaced with aserine, wherein the humanized CC49 antibody retains binding affinity forTAG-72 and has reduced immunogenicity, when compared to a parentalhumanized CC49 antibody.
 16. The recombinant nucleic acid sequence ofclaim 15, wherein at least one amino acid of positions 60, 61, 62, or 64in the murine CC49 H-CDR2 is replaced with an amino acid at acorresponding position in the human monoclonal 21/28′CL antibody. 17.The recombinant nucleic acid sequence of claim 16, wherein an asparagineat position 60 in the murine CC49 H-CDR2 is replaced with a serine, aglutamic acid at position 61 in the murine CC49 H-CDR2 is replaced witha glutamine, an arginine at position 62 in the murine CC49 H-CDR2 isreplaced with a lysine, or a lysine at position 64 in the murine CC49H-CDR2 is replaced with a glutamine.
 18. The recombinant nucleic acidsequence of claim 15, wherein the parental humanized CC49 antibodycomprises three L-CDRs and three H-CDRs of the murine monoclonal CC49antibody, a variable light chain framework of a human monoclonal LENantibody, and a variable heavy chain framework of a human monoclonal21/28′CL antibody.
 19. A recombinant nucleic acid sequence comprising apromoter sequence operably linked to the recombinant nucleic acidsequence of claim
 16. 20. A recombinant vector comprising therecombinant nucleic acid sequence of claim
 19. 21. A host celltransformed with the recombinant vector of claim
 20. 22. A recombinantnucleic acid sequence encoding a humanized anti-TAG-72 CC49 antibody,wherein the humanized anti-TAG-72 antibody comprises: a light chainComplementarity Determining Region (L-CDR)1, a L-CDR2, and a L-CDR3; anda heavy chain Complementarity Determining (H-CDR)1, a H-CDR2, and aH-CDR3, wherein L-CDR3, H-CDR1, H-CDR2 and H-CDR3 are of a murine CC49antibody and L-CDR1 and L-CDR2 are of a human monoclonal LEN antibodyL-CDR1 and L-CDR2 respectively, and wherein (1) a threonine is atposition 94 in the L-CDR3, or (2) a serine is at position 97 in theL-CDR3, or (3) a threonine is at position 94 and a serine is at position97 in the L-CDR3, wherein the humanized CC49 antibody retains bindingaffinity for TAG-72 and has reduced immunogenicity, when compared to aparental humanized CC49 antibody.
 23. The nucleic acid sequence of claim22, wherein the parental humanized CC49 antibody comprises three L-CDRsand three H-CDRs of the murine monoclonal CC49 antibody, a variablelight chain framework of a human monoclonal LEN antibody, and a variableheavy chain framework of a human monoclonal 21/28′CL antibody.
 24. Arecombinant nucleic acid sequence comprising a promoter sequenceoperably linked to the recombinant nucleic acid sequence of claim 22.25. A recombinant vector comprising the recombinant nucleic acidsequence of claim
 22. 26. A host cell transformed with the recombinantvector of claim 25.